Production of high quality durum wheat having increased amylose content

ABSTRACT

The present invention provides compositions and methods of altering/improving Durum wheat phenotypes. Furthermore, methods of breeding Durum wheat and/or other closely related species to produce plants having altered or improved phenotypes are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No.61/736,136 filed on Dec. 12, 2012, and U.S. provisional application No.61/717,357 filed on Oct. 23, 2012, both of which are hereby incorporatedby reference in their entirety for all purposes.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:MONT_135_01WO_Seq_List.txt, date recorded: Oct. 10, 2013; file size: 136kilobytes).

TECHNICAL FIELD

The invention generally relates to improving the end product qualitycharacteristics of durum wheat. More specifically, the present inventionrelates to compositions and methods for improving one or more endproduct quality characteristics of wheat by modifying one or more starchsynthesis genes.

BACKGROUND

Starch makes up approximately 70% of the dry weight of cereal grains andis composed of two forms of glucose polymers, straight chained amylosewith α-1,4 linkages and branched amylopectin with α-1,4 linkages andα-1,6 branch points. In bread wheat, amylose accounts for approximately25% of the starch with amylopectin the other 75% (reviewed in Tetlow2006). The synthesis of starch granules is an intricate process thatinvolves several enzymes which associate in complexes (Tetlow et al.2008; Tetlow et al. 2004b). In bread wheat, the “waxy” proteins (granulebound starch synthase 1) encoded by the genes Wx-A1a, Wx-B1a, and Wx-D1aare solely responsible for amylose synthesis after the production ofADP-glucose by ADP-glucose pyrophosphorylase (AGPase) (Denyer et al.1995; Miura et al. 1994; Yamamori et al. 1994). In contrast, amylopectinsynthesis involves a host of enzymes such as AGPase, starch synthases(SS) I, II, III, IV, starch branching enzymes (SBE) I and II, and starchde-branching enzymes (Tetlow et al. 2004a).

The majority of durum wheat is used for pasta and pasta products, butthere is interest in investigating durum wheat for noodle production.There are several reasons for interest in durum noodle production.First, it would provide an additional market for durum wheat grain.Durum wheat is lower than bread wheat in polyphenol oxidase, an enzymecausing noodles to turn gray or brown with time. Finally, the high levelof carotenoids present in durum wheat could produce enhanced yellowcolor for alkaline noodles. The proportion of amylose to amylopectin isan important factor in determining end product properties in durumwheat. Much attention has been devoted to determining the impacts ofreduced amylose on Asian noodle quality in bread wheat. Information islacking on the impacts of small increases in amylose on end productquality in durum wheat. Therefore, there is a great need in compositionsand methods of modifying amylose in durum wheat. The present inventionprovides compositions and methods for producing improved durum wheatplants through conventional plant breeding and/or molecularmethodologies.

SUMMARY OF INVENTION

The present invention provides for high amylose durum wheat grain. Insome embodiments, the grain is produced from a durum wheat plant of thepresent invention. In some embodiments, the grain is produced from adurum wheat comprising one or more mutations of one or more starchsynthesis genes. In some embodiments, the grain is produced from a durumwheat comprising one or more mutations of a durum starch granuleprotein-B1 (SGP-B1) gene. In some embodiments, the present invention isa high amylose grain produced from a durum wheat plant comprising one ormore mutations of a durum starch granule protein-B1 (SGP-B1) gene of awild type durum wheat plant, wherein the amylose content in said highamylose grain is increased when compared to grain of a wild type durumwheat plant grown at the same time under similar field conditions. Insome embodiments, the wheat grain is produced from a durum wheatcomprising one or more mutations of a durum starch granule protein-B1(SGP-B1) gene, and one or more mutations of a durum starch granuleprotein-A1 (SGP-A1) gene. In some embodiments, the proportion of amylosecontent in the starch of the grain is at least 40% as measured bydifferential scanning calorimetry analysis. In other embodiments theamylose content of the starch grain is at least 50%. In someembodiments, the amylose content in the starch of said high amylosegrain is increased when compared to the starch of a grain of anappropriate durum wheat check variety grown under similar fieldconditions. In some embodiments, the durum wheat check variety is grownat the same time as the high amylose durum wheat plant.

In some embodiments, the one or more mutations are selected from a groupconsisting of a mutation of a starch granule protein-A1 (SGP-A1) alleleof a wild type durum wheat plant and/or a mutation of a starch granuleprotein-B1 (SGP-B1) allele of a wild type durum wheat plant. In someembodiments, the one or more mutations of the high amylose graincomprise a deletion in the first exon of the SGP-A1 gene. In someembodiments, the deletion is at nucleotide position 145-174 of theSGP-A1 gene. In some embodiments, the one or more mutations of the highamylose grain comprise a nucleotide substitution at nucleotide position979 and/or position 1864 of the SGP-B1 gene. In some embodiments, theone or more genetic mutations comprise null mutations for at least oneSGP-A1 gene and/or at least one SGP-B1 gene. In some embodiments, theSGP-B1 mutation leads to an amino acid substitution from aspartic acidto asparagines at amino acid position 327 of SGP-B1, and/or an aminoacid substitution from aspartic acid to asparagines at amino acidposition 622 of SGP-B1. In some embodiments, the mutation of the SGP-A1allele or the SGP-B1 allele is caused by artificial mutagenesis ornatural mutation. In some embodiments, the mutation is caused bynucleotide substitution, insertion, deletion, and/or genomere-arrangement.

The present invention also discloses the plant cells of high amylosewheat. In some embodiments, the plant cells include cells from any plantpart such as plant protoplasts, plant cell tissue cultures from whichwheat plants can be regenerated, plant calli, embryos, pollen, grain,ovules, fruit, flowers, leaves, seeds, roots, root tips and the like.

Other embodiments of the present invention include flour based productsfrom durum wheat grain produced from a durum wheat comprising one ormore mutations of a durum starch granule protein-B1 (SGP-B1) gene, andone or more mutations of a durum starch granule protein-A1 (SGP-A1)gene. In some embodiments, the high amylose grain can be used to produceflour based products. In some embodiments, milled products produced fromthe high amylose grain are flour, starch, semolina, among others. Insome embodiments, flour based products produced from the high amylosegrain are pasta, and noodles among others. The present invention teachesflour based products produced from the high amylose grain. In someembodiments, the invention teaches flour produced from the high amylosegrain. In other embodiments the flour based product produced by the highamylose grain is dried pasta. In some embodiments, the flour basedproduct has a protein content of at least 17%. In other embodiments theflour based product has a protein content of at least 20%. In someembodiments, the flour based product has a dietary fiber content of atleast 3%. In other embodiments the flour based product has a dietaryfiber content of at least 7%. In some embodiments, the flour basedproduct has a resistant starch content of at least 2%. In otherembodiments the flour based product has a resistant starch content of atleast 3%. In other embodiments the protein, resistant starch and dietaryfiber contents of the flour based product are increased when compared toa flour based product from an appropriate durum wheat check line grownunder similar field conditions. In some embodiments, of the presentinvention, when the comparison is to an appropriate durum wheat checkline grown under similar field conditions, the wheat lines of thepresent invention and then check lines are grown at the same time and/orlocation. For example, in some embodiments, the flour based product hasan increased protein content that is at least 10% higher than a flourbased product produced from the grain of an appropriate durum wheatcheck variety grown under similar field conditions. In other embodimentsthe flour based product has an increased protein content that is atleast 20% higher than a flour based product produced from the grain ofan appropriate durum wheat check variety grown under similar fieldconditions. In other embodiments the flour based product has anincreased protein content that is at least 30% higher than a flour basedproduct produced from the grain of an appropriate durum wheat checkvariety grown under similar field conditions. In some embodiments, theflour based product has an increased dietary fiber content that is atleast 50% higher than a flour based product produced from the grain ofan appropriate durum wheat check variety grown under similar fieldconditions. In other embodiments the flour based product has anincreased dietary fiber content that is at least 100% higher than aflour based product produced from the grain of an appropriate durumwheat check variety grown under similar field conditions. In otherembodiments the flour based product has an increased dietary fibercontent that is at least 200% higher than a flour based product producedfrom the grain of an appropriate durum wheat check variety grown undersimilar field conditions. In some embodiments, the flour based producthas an increased resistant starch content that is at least 50% higherthan a flour based product produced from the grain of an appropriatedurum wheat check variety grown under similar field conditions. In otherembodiments the flour based product has an increased resistant starchcontent that is at least 100% higher than a flour based product producedfrom the grain of an appropriate durum wheat check variety grown undersimilar field conditions. In other embodiments the flour based producthas an increased resistant starch content that is at least 200% higherthan a flour based product produced from the grain of an appropriatedurum wheat check variety grown under similar field conditions. In someembodiments, the flour based product has an increased amylose contentthat is at least 12% higher than a flour based product produced from thegrain of an appropriate durum wheat check variety grown under similarfield conditions. In other embodiments the flour based product has anincreased amylose content that is at least 25% higher than a flour basedproduct produced from the grain of an appropriate durum wheat checkvariety grown under similar field conditions. In other embodiments theflour based product has an increased amylose content that is at least40% higher than a flour based product produced from the grain of anappropriate durum wheat check variety grown under similar fieldconditions. In some embodiments, the flour based product is dried pastawherein the pasta has improved firmness after cooking compared to pastaproduced from the grain of an appropriate durum wheat check varietygrown under similar field conditions.

In some embodiments, the high amylose grain has a flour swelling power(FSP) of less than 8.4. In other embodiments the high amylose grain hasan FSP of less than 7.5.

In some embodiments, the proportion of dietary fiber, resistant starch,and protein content that is increased in said high amylose grain isincreased when compared to the grain of an appropriate durum wheat checkvariety grown under similar field conditions. In some embodiments, theamylose content of the starch made from the high amylose grain is atleast 12% higher than the amylose content of the starch made from thegrain of an appropriate wheat check variety grown under similar fieldconditions. In other embodiments, the amylose content of the starch madefrom the high amylose grain is at least 25% higher than the amylosecontent of the starch made from the grain of an appropriate wheat checkvariety grown under similar field conditions. In other embodiments, theamylose content of the starch made from the high amylose grain is atleast 40% higher than the amylose content of the starch made from thegrain of an appropriate wheat check variety grown under similar fieldconditions. In some embodiments, the appropriate durum wheat checkvariety is grown at the same time and/or location.

In some embodiments, the starch of the high amylose grain has alteredgelatinization properties when compared to starch from the grain of anappropriate durum wheat check variety grown under similar fieldconditions.

In some embodiments, the pasta or noodles made from the high amylosegrain have reduced glycemic index compared to pasta or noodles producedfrom the grain of an appropriate durum wheat check variety grown undersimilar field conditions.

In some embodiments, the pasta or noodles made from the high amylosegrain have increased firmness compared to pasta or noodles made fromgrain of the appropriate durum wheat check variety grown under similarfield conditions.

In some embodiments, the pasta or noodles made from the high amylosegrain have increased tolerance to overcooking compared to pasta ornoodles made from grain of the appropriate durum wheat check varietygrown under similar field conditions.

In some embodiments, the pasta or noodles made from the high amylosegrain have increased protein content compared to pasta or noodles madefrom grain of the appropriate durum wheat check variety grown undersimilar field conditions.

Pasta produced from the mutant grain also has increased proportion ofdietary fiber, resistant starch and/or protein content when compared topasta made from the grain of the wild type durum wheat plant.

In some embodiments, the grain has increased amylose content compared tothe grain of the wild type durum wheat plant.

In some embodiments, the grain has increased dietary fiber and increasedamylose content when compared to the grain of the wild type durum wheatplant.

In some embodiments, the grain has increased protein content andincreased amylose content when compared to the grain of the wild typedurum wheat plant.

In some embodiments, the grain has increased dietary fiber and decreasedendosperm to bran ratio and/or reduced milling yield when compared tothe grain of the wild type durum wheat plant.

In some embodiments, the grain has increased dietary fiber and increasedash when compared to the grain of the wild type durum wheat plant.

In some embodiments, the grain has increased protein and reduced starchcontent when compared to the grain of the wild type durum wheat plant.

In some embodiments, the mutant durum wheat starch has an increasedamylose content when compared to the wild type durum wheat starch. Insome embodiments, the amylose content of the mutant durum wheat is about38% to about 50%.

In some embodiments, the starch of the present invention has an overalldecrease in the amount of B-type starch granules when compared to starchthe of an appropriate wheat check variety grown under similar fieldconditions.

In some embodiments, the starch of the present invention has an alteredgelatinization property when compared to the wild type durum wheatstarch.

In some embodiments, the grain produced imparts increased firmness tofood, such as pasta or noodles produced from the durum wheat plant whencompared to food, such as pasta or noodles produced from the wild typedurum wheat plant.

In some embodiments, the grain of the present invention imparts reducedglycemic index to pasta or noodles produced from the durum wheat plantwhen compared to pasta or noodles produced from the wild type durumwheat plant.

In some embodiments, the grain of the present invention has increasedtolerance to overcooking when compared to the wild type durum wheatstarch.

The present invention also provides flour produced from the grain of thepresent invention.

The present invention also provides starch produced from the grain ofthe present invention.

The present invention also provides methods for producing a high amylosedurum wheat plant. In some embodiments, the methods comprise performingmutagenesis on durum wheat plant that comprises a SGP-A1 mutation and/ora SGP-B1 mutation. In some embodiments, the durum wheat plant comprisesa SGP-A1 with a 29 bp deletion in the first exon. In some embodiments,the durum wheat plant comprises a SGP-B1 with amino acid substitutionfrom at amino acid position 327 of SGP-B1, e.g., from aspartic acid toasparagines, and/or an amino acid substitution at amino acid position622 of SGP-B1, e.g., from aspartic acid to asparagines. The methodsproduce a durum wheat plant with an elevated amylose content whencompared to a wild type durum wheat plant.

-   -   The present invention also provides methods for producing durum        wheat with one or more mutations of a durum starch granule        protein (SGP-B1). In some embodiments, the invention provides        methods for producing durum wheat with one or more mutations of        a durum starch granule protein (SGP-B1), and one or more        mutations of a durum starch granule protein-A1 (SGP-A1) gene. In        some embodiments, the method comprises mutagenizing a durum        wheat grain containing one or more mutations of a durum starch        granule protein-A1 (SGP-A1) gene to form a mutagenized        population of grain; growing one or more durum wheat plants from        said mutagenized durum wheat grain; screening the resulting        plants to identify durum wheat plants with a durum SGP-B1 mutant        gene; and, selecting one or more durum wheat plants containing        the durum SGP-B1 mutant gene. In other embodiments the method        comprises mutagenizing a durum wheat grain containing one or        more mutations of a durum starch granule protein-B1 (SGP-B1)        gene to form a mutagenized population of grain; growing one or        more durum wheat plants from said mutagenized durum wheat grain;        screening the resulting plants to identify durum wheat plants        with a durum SGP-A1 mutant gene; and, selecting one or more        durum wheat plants containing the durum SGP-A1 mutant gene. In        some embodiments, the resulting durum wheat plant comprises one        or more mutations of a durum starch granule protein-B1 (SGP-B1)        gene, and one or more mutations of a durum starch granule        protein-A1 (SGP-A1) gene, and wherein said plant produces high        amylose grain. In other embodiments the method for producing the        durum wheat plant with one or more mutations of a durum starch        granule protein (SGP-B1), and one or more mutations of a durum        starch granule protein-A1 (SGP-A1) gene comprises crossing a        durum wheat plant containing one or more mutations on a durum        SGP-A1 gene with a second durum wheat plant containing one or        more mutations on a durum SGP-B1 gene; harvesting the resulting        seed; and, growing the harvested seed. In some embodiments, the        resulting durum wheat plant comprises one or more mutations of a        durum starch granule protein-B1 (SGP-B1) gene, and one or more        mutations of a durum starch granule protein-A1 (SGP-A1) gene,        and wherein said plant produces high amylose grain.

The present invention also provides methods for culturing plant tissue.In some embodiments, the method of culturing and regenerating planttissue comprises culturing at least part of the high amylose durum wheatplant in conditions conducive to plant regeneration, therebyregenerating said plant. The present invention also provides methods ofproducing hybrid seeds, the method comprising crossing the high amylosedurum wheat with another plant, and harvesting the resultant seed. Thepresent invention also provides methods of breeding durum wheat plantswith high amylose grain comprising making a cross between a first highamylose durum plant with a second plant to produce a F1 plant;backcrossing the F1 plant to the second plant; and repeating thebackcrossing step one or more times to generate a near isogenic orisogenic line. In some embodiments, the resulting plant has the SGP-A1and SGP-B1 mutations integrated into the genome of the second plant andthe near isogenic or isogenic line derived from the second plant withthe SGP-A1 and/or SBP-B1 mutations.

The present invention also provides methods for increasing firmness in afood product produced from durum wheat grain. In some embodiments, thefood product is noodle or pasta. In some embodiments, the methodscomprise producing the noodle or pasta from a durum wheat plant whereinsaid durum wheat plant includes at least one mutation in the SGP-Iprotein. The durum wheat plant produces grain with an elevated amylosecontent when compared to a wild type durum wheat plant. In someembodiments, the food product produced from such durum wheat plant ismore resistant to overcooking compared to food product produced fromgrain of a wild-type durum wheat plant. In some embodiments, at leastone mutation is selected from a group consisting of a mutation of astarch granule protein-A1 (SGP-A1) allele and a mutation of a starchgranule protein-B1 (SGP-B1) allele.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts SDS-PAGE analysis of starch granule proteins fromMountrail (SSIIa-Aa) and PI 330546 (SSIIa-Ab) and segregatingrecombinant inbred lines from their cross.

FIG. 2 depicts the relationship between flour swelling power and noodlefirmness for recombinant inbred lines from Mountrail/PI 330546 andMountrail/IG 86304 where both crosses are segregating for SSIIa-Aaversus SSIIa-Ab. Response equations are: IG 86304 SSIIa-Aaŷ=10.489−0.054x±0.029; IG 86304 SSIIa-Ab ŷ=8.324 1 0.018x±0.057; PI330546 SSIIa-Aa ŷ10.671−0.060x±0.026; and PI 330546 SSIIa-Abŷ=10.080-0.069±0.026.

FIG. 3 depicts SDS-PAGE analysis of starch granule proteins fromMountrail/PI-330546 F₅ SGP-1 wild-type (WT), Mountrail/PI-330546 F₅SGP-A1 null (A null) and SGP-1 double null genotypes DHA175 and DHA55.The acrylamide gel was silver stained and a dilution series of WT wasused to create the loading curve. The elimination of both SGP-1 proteinsin durum results in reduced binding of SGP-2 and SGP-3.

FIG. 4 depicts FEM micrograph of starch granules fromMountrail/PI-330546 F5 (SGP-1 wild-type), Mountrail/PI-330546 F5 (SGP-A1null) and SGP-1 double null genotype DHA175.

FIG. 5 depicts DSC thermogram of starches from Mountrail/PI-330546 F₅SGP-1 wild-type, Mountrail/PI-330546 F₅ SGP-A1 null and SGP-1 doublenull genotypes DHA175 and DHA55. Approximately 10 mg of starch (actualweight was recorded) per sample was placed in a high-pressure stainlesssteel pan along with 55 μL of ddH2O. The pan was sealed with an O-ringand cover and the starch was left to hydrate overnight at roomtemperature. Samples were re-weighed the next day then placed at 25° C.for two min to equilibrate before they were heated to 120° C. at 10°C./min. Heat transfer in the samples was compared to an empty stainlesssteel pan as a reference. The Pyris software was used to generatethermograms and calculate transition temperatures and heat of physicaltransition. Amylose was determined via DSC using the methods describedin Polaske et al. (2005). Statistical analysis on amylose content wascarried out using PROC GLM and t-tests with an alpha of 0.05 in SAS 9.0(SAS Institute, Cary, N.C.). SGP-1 double null lines show an alteredamylopectin gelatinization profile that occurs at cooler temperaturesand has decreased enthalpy compared to the wild-type and SGP-A1 nullcontrols.

FIG. 6 depicts the glycemic index for DHA175 and wild-type control wheatpastas. The glycemic index was determined by calculating the incrementalarea under the two-hour blood glucose response curve (AUC) following a12-hour fast and ingestion of DHA175 or wild-type durum pasta. DHA175durum wheat pasta exhibits a lower glycemic index than wild-type pasta.

FIG. 7 depicts plasma glucose curves over the course of 120 minutesfollowing a 12-hour fast and ingestion of DHA175 or wild-type durumpasta. DHA175 pasta also exhibited plasma glucose curves with lowerglucose peaks and higher sustained glucose levels at 90 and 120 minuteswhen compared to wild time control durum.

SEQUENCES

Sequence listings for SEQ ID No: 1-SEQ ID No: 24 are part of thisapplication and are incorporated by reference herein. Sequence listingsare provided at the end of this document.

DETAILED DESCRIPTION

All publications, patents and patent applications, including anydrawings and appendices, and all nucleic acid sequences and polypeptidesequences identified by GenBank Accession numbers, herein areincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

Definitions

As used herein, the verb “comprise” as is used in this description andin the claims and its conjugations are used in its non-limiting sense tomean that items following the word are included, but items notspecifically mentioned are not excluded.

The invention provides compositions and methods for improving the endproduct quality characteristics of plants. As used herein, the term“plant” refers to wheat (e.g., bread wheat or durum wheat), unlessspecified otherwise.

As used herein, the term “plant” also includes the whole plant or anyparts or derivatives thereof, such as plant cells, plant protoplasts,plant cell tissue cultures from which wheat plants can be regenerated,plant calli, embryos, pollen, grain, ovules, fruit, flowers, leaves,seeds, roots, root tips and the like.

As used herein, the term “appropriate durum wheat check”, is meant torepresent a durum wheat plant which provides a basis for evaluation ofthe experimental plants of the present invention. An appropriate checkis grown under the same environmental conditions, as is the experimentalline, and is of approximately the same maturity as the experimentalline. The term “appropriate durum wheat check” may actually reflectmultiple appropriate varieties chosen to represent control lines for themodification or factor being tested in the experimental line. In someembodiments, the appropriate durum wheat check variety can be a wildtype durum wheat variety without the experimental mutation. In someembodiments, durum wheat check lines can be ‘Mountrail’, ‘Divide’,‘Strongfield’, or ‘Alazda’ wild type varieties.

The invention provides plant parts. As used herein, the term “plantpart” refers to any part of a plant including but not limited to theshoot, root, stem, seeds, stipules, leaves, petals, flowers, ovules,bracts, branches, petioles, internodes, bark, pubescence, tillers,rhizomes, fronds, blades, pollen, stamen, plant cells, and the like.

The term “a” or “an” refers to one or more of that entity; for example,“a gene” refers to one or more genes or at least one gene. As such, theterms “a” (or “an”), “one or more” and “at least one” are usedinterchangeably herein. In addition, reference to “an element” by theindefinite article “a” or “an” does not exclude the possibility thatmore than one of the elements are present, unless the context clearlyrequires that there is one and only one of the elements.

The invention provides selectable marker. As used herein, the phrase“plant selectable or screenable marker” refers to a genetic markerfunctional in a plant cell. A selectable marker allows cells containingand expressing that marker to grow under conditions unfavorable togrowth of cells not expressing that marker. A screenable markerfacilitates identification of cells which express that marker.

The invention provides inbred plants. As used herein, the terms “inbred”and “inbred plant” are used in the context of the present invention.This also includes any single gene conversions of that inbred.

The term “single allele converted plant” as used herein refers to thoseplants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of an inbred are recovered in addition tothe single allele transferred into the inbred via the backcrossingtechnique.

The invention provides plant samples. As used herein, the term “sample”includes a sample from a plant, a plant part, a plant cell, or from atransmission vector, or a soil, water or air sample.

The invention provides plant offsprings. As used herein, the term“offspring” refers to any plant resulting as progeny from a vegetativeor sexual reproduction from one or more parent plants or descendantsthereof. For instance an offspring plant may be obtained by cloning orselfing of a parent plant or by crossing two parent plants and includeselfings as well as the F1 or F2 or still further generations. An F1 isa first-generation offspring produced from parents at least one of whichis used for the first time as donor of a trait, while offspring ofsecond generation (F2) or subsequent generations (F3, F4, etc.) arespecimens produced from selfings of F1's, F2's etc. An F1 may thus be(and usually is) a hybrid resulting from a cross between two truebreeding parents (true-breeding is homozygous for a trait), while an F2may be (and usually is) an offspring resulting from self-pollination ofsaid F1 hybrids.

The invention provides methods for crossing a first plant comprisingrecombinant sequences with a second plant. As used herein, the term“cross”, “crossing”, “cross pollination” or “cross-breeding” refer tothe process by which the pollen of one flower on one plant is applied(artificially or naturally) to the ovule (stigma) of a flower on anotherplant.

The invention provides plant cultivars. As used herein, the term“cultivar” refers to a variety, strain or race of plant that has beenproduced by horticultural or agronomic techniques and is not normallyfound in wild populations.

The invention provides plant genes. As used herein, the term “gene”refers to any segment of DNA associated with a biological function.Thus, genes include, but are not limited to, coding sequences and/or theregulatory sequences required for their expression. Genes can alsoinclude nonexpressed DNA segments that, for example, form recognitionsequences for other proteins. Genes can be obtained from a variety ofsources, including cloning from a source of interest or synthesizingfrom known or predicted sequence information, and may include sequencesdesigned to have desired parameters.

The invention provides plant genotypes. As used herein, the term“genotype” refers to the genetic makeup of an individual cell, cellculture, tissue, organism (e.g., a plant), or group of organisms.

In some embodiments, the present invention provides homozygotes ofplants. As used herein, the term “hemizygous” refers to a cell, tissueor organism in which a gene is present only once in a genotype, as agene in a haploid cell or organism, a sex-linked gene in theheterogametic sex, or a gene in a segment of chromosome in a diploidcell or organism where its partner segment has been deleted.

In some embodiments, the present invention provides heterologous nucleicacids. As used herein, the terms “heterologous polynucleotide” or a“heterologous nucleic acid” or an “exogenous DNA segment” refer to apolynucleotide, nucleic acid or DNA segment that originates from asource foreign to the particular host cell, or, if from the same source,is modified from its original form. Thus, a heterologous gene in a hostcell includes a gene that is endogenous to the particular host cell, buthas been modified. Thus, the terms refer to a DNA segment which isforeign or heterologous to the cell, or homologous to the cell but in aposition within the host cell nucleic acid in which the element is notordinarily found. Exogenous DNA segments are expressed to yieldexogenous polypeptides.

In some embodiments, the present invention provides heterologous traits.As used herein, the term “heterologous trait” refers to a phenotypeimparted to a transformed host cell or transgenic organism by anexogenous DNA segment, heterologous polynucleotide or heterologousnucleic acid.

In some embodiments, the present invention provides heterozygotes. Asused herein, the term “heterozygote” refers to a diploid or polyploidindividual cell or plant having different alleles (forms of a givengene) present at least at one locus.

In some embodiments, the present invention provides heterozygous traits.As used herein, the term “heterozygous” refers to the presence ofdifferent alleles (forms of a given gene) at a particular gene locus.

In some embodiments, the present invention provides homologs. As usedherein, the terms “homolog” or “homologue” refer to a nucleic acid orpeptide sequence which has a common origin and functions similarly to anucleic acid or peptide sequence from another species.

In some embodiments, the present invention provides homozygotes. As usedherein, the term “homozygote” refers to an individual cell or planthaving the same alleles at one or more or all loci. When the term isused with reference to a specific locus or gene, it means at least thatlocus or gene has the same alleles.

In some embodiments, the present invention provides homozygous traits.As used herein, the terms “homozygous” or “HOMO” refer to the presenceof identical alleles at one or more or all loci in homologouschromosomal segments. When the terms are used with reference to aspecific locus or gene, it means at least that locus or gene has thesame alleles.

In some embodiments, the present invention provides hybrids. As usedherein, the term “hybrid” refers to any individual cell, tissue or plantresulting from a cross between parents that differ in one or more genes.

In some embodiments, the present invention provides mutants. As usedherein, the terms “mutant” or “mutation” refer to a gene, cell, ororganism with an abnormal genetic constitution that may result in avariant phenotype.

The invention provides open-pollinated populations. As used herein, theterms “open-pollinated population” or “open-pollinated variety” refer toplants normally capable of at least some cross-fertilization, selectedto a standard, that may show variation but that also have one or moregenotypic or phenotypic characteristics by which the population or thevariety can be differentiated from others. A hybrid, which has nobarriers to cross-pollination, is an open-pollinated population or anopen-pollinated variety.

The invention provides plant ovules and pollens. As used herein whendiscussing plants, the term “ovule” refers to the female gametophyte,whereas the term “pollen” means the male gametophyte.

The invention provides plant phenotypes. As used herein, the term“phenotype” refers to the observable characters of an individual cell,cell culture, organism (e.g., a plant), or group of organisms whichresults from the interaction between that individual's genetic makeup(i.e., genotype) and the environment.

The invention provides plant tissue. As used herein, the term “planttissue” refers to any part of a plant. Examples of plant organs include,but are not limited to the leaf, stem, root, tuber, seed, branch,pubescence, nodule, leaf axil, flower, pollen, stamen, pistil, petal,peduncle, stalk, stigma, style, bract, fruit, trunk, carpel, sepal,anther, ovule, pedicel, needle, cone, rhizome, stolon, shoot, pericarp,endosperm, placenta, berry, stamen, and leaf sheath.

The invention provides self-pollination populations. As used herein, theterm “self-crossing”, “self pollinated” or “self-pollination” means thepollen of one flower on one plant is applied (artificially or naturally)to the ovule (stigma) of the same or a different flower on the sameplant.

As used herein, the term “amylose content” refers to the amount ofamylose in wheat starch. Amylose is a linear polymer of α-1,4 linkedD-glucose with relatively few side chains. Amylose is digested moreslowly than amylopectin which while also having linear polymers of α-1,4linked D-glucose has many α-1,6 D-glucose side chains. Amylose absorbsless water upon heating than amylopectin and is digested more slowly.Amylose content can be measured by colormetric assays involvingiodine-potassium iodide assays, by DSC, Con A, or estimated by measuringthe water absorbing capacity of flour or starch after heating.

As used herein, the term “starch synthesis genes” refers to any genesthat directly or indirectly contribute to, regulate, or affect starchsynthesis in a plant. Such genes includes, but are not limited to genesencoding waxy protein (a.k.a., Granule bound starch synthases (GBSS),such as GBSSI, GBSSII), ADP-glucose pyrophosphorylases (AGPases), starchbranching enzymes (a.k.a., SBE, such as SBE I and SBE II), starchde-branching enzymes (a.k.a., SDBE), and starch synthases I, II, III,and IV.

As used herein, the term “waxy protein”, “Granule bound starchsynthase”, GBSS, or “ADP-glucose:(1->4)-alpha-D-glucan4-alpha-D-glucosyltransferase” refers to a protein having E.C. number2.4.1.21, which can catalyze the following reaction:

ADP-glucose+(1,4-alpha-D-glucosyl)n=ADP+(1,4-alpha-D-glucosyl)n+1

As used herein, the term “ADP-glucose pyrophosphorylase”, AGPase,“adenosine diphosphate glucose pyrophosphorylase”, or“adenosine-5′-diphosphoglucose pyrophosphorylase” refers to a proteinhaving E.C. number 2.7.7.27, which can catalyze the following reaction:

ATP+alpha-D-glucose 1-phosphate=diphosphate+ADP-glucose

As used herein, the term “starch branching enzyme”, SBE, “branchingenzyme”, BE, “glycogen branching enzyme”, “1,4-alpha-glucan branchingenzyme”, “alpha-1,4-glucan:alpha-1,4-glucan 6-glycosyltransferase” or“(1->4)-alpha-D-glucan:(1->4)-alpha-D-glucan6-alpha-D-[(1->4)-alpha-D-glucano]-transferase” refers to a proteinhaving E.C. number 2.4.1.18, which can catalyze the following reaction:

2 1,4-alpha-D-glucan=alpha-1,4-D-glucan-alpha-1,6-(alpha-1,4-D-glucan)

As used herein, the term “starch de-branching enzymes”, SDBE, orisoamylase refers to a protein having the E.C. number 2.4.1.1, 2.4.1.25,3.2.1.68 or 3.2.1.41, which can hydrolyse alpha-1,6 glucosidic bonds inglucans containing both alpha-1,4 and alpha-1,6 linkages.

As used herein, the term starch synthase I, II, III, or IV (SSI or SI,SSII or SII, SSIII or SOOO, and SSIV or SIV), refers to a protein ofstarch synthase class I, class II, class III, or class IV, respectively.Such as protein that is involved in amylopectin synthesis.

As used herein, the term starch granule protein-1 or SGP-1 refers to aprotein belonging to starch synthase class 11, contained in wheat starchgranules (Yamamori and Endo, 1996).

As used herein, the term wheat refers to any wheat species within thegenus of Triticum, or the tribe of Triticeae, which includes, but arenot limited to, diploid, tetraploid, and hexaploid wheat species.

As used herein, the term “milled product” refers to a product producedfrom grinding grains (from wheat or other grain producing plants).Non-limiting examples of milled products include: flour, all purposeflour, starch, bread flour, cake flour, self-rising flour, pastry flour,semolina, durum flour, whole wheat flour, stone ground flour, glutenflour, and graham flour among others.

As used herein, the term “flour based product” refers to products madefrom flour including: pasta, noodles, bread products, cookies, andpastries among others.

As used herein, the term “high amylose grain” refers to a durum wheatgrain with starch with high levels of amylose. In some embodiments, thehigh amylose levels are elevated compared to the amylose content of awheat grain from a wild type or other appropriate durum wheat checkvariety grown at the same time under similar field conditions. In otherembodiments the amylose levels are high in absolute percentage terms asmeasured by differential scanning calorimetry analysis.

As used herein, the term diploid wheat refers to wheat species that havetwo homologous copies of each chromosome, such as Einkorn wheat (T.monococcum), having the genome AA.

As used herein, the term tetraploid wheat refers to wheat species thathave four homologous copies of each chromosome, such as emmer and durumwheat, which are derived from wild emmer (T. dicoccoides). Wild emmer isitself the result of a hybridization between two diploid wild grasses,T. urartu and a wild goatgrass such as Aegilops searsii or Ae.speltoides. The hybridization that formed wild emmer (having genomeAABB) occurred in the wild, long before domestication, and was driven bynatural selection.

As used herein, the term hexaploid wheat refers to wheat species thathave six homologous copies of each chromosome, such as bread wheat.Either domesticated emmer or durum wheat hybridized with another wilddiploid grass (Aegilops tauschii, having genome DD) to make thehexaploid wheats (having genome AABBDD).

As used herein, SSIIa-Aa refers to both wild type “aa” alleles beingpresent but SSIIa-Ab refers to both “bb” alleles being present. SSIIaand SSIIb would be two different forms of the same enzyme.

As used herein, the term “gelatinization temperature” refers to thetemperature at which starch is dissolved in water during heating.Gelatinization temperature is related to amylose content with increasedamylose content associated with increased gelatinization temperature.

As used herein, the term “starch retrogradation” refers to the firmnessof starch water gels with increased amylose associated with increasedstarch retrogradation and firmer starch based gels.

As used herein, the term “flour swelling power” or FSP refers to theweight of flour or starch based gel relative to the weight of theoriginal sample after heating in the presence of excess water. Increasedamylose is associated with decreased FSP.

As used herein, the term “grain hardness” refers to the pressurerequired to fracture grains and is related to particle size aftermilling, milling yield, and some end product quality traits. Increasedgrain hardness is associated with increased flour particle size,increased starch damage and decreased break flour yield.

As used herein, the term “semolina” refers to the coarse, purified wheatmiddlings of durum wheat.

As used herein, the term “resistant amylose” refers to amylose whichresists digestion and thus serves a purpose in the manufacturing ofreduced glycemic index food products.

As used herein, the term “resistant starch” refers to starch thatresists digestion and behaves like dietary fiber. Increased amylose isbelieved to be associated with increased resistant starch.

As used herein, the term “allele” refers to any of several alternativeforms of a gene.

As used herein, “starch” refers to starch in its natural or native formas well as also referring to starch modified by physical, chemical,enzymatic and biological processes.

As used herein. “amylose” refers to a starch polymer that is anessentially linear assemblage of D-anhydroglucose units which are linkedby alpha 1,6-D-glucosidic bonds.

As used herein, “amylose content” refers to the percentage of theamylose type polymer in relation to other starch polymers such asamylopectin.

As used herein, the term “grain” refers to mature wheat kernels producedby commercial growers for purposes other than growing or reproducing thespecies.

As used herein, the term “kernel” refers to the wheat caryopsiscomprising a mature embryo and endosperm which are products of doublefertilization.

As used herein, the term “line” is used broadly to include, but is notlimited to, a group of plants vegetatively propagated from a singleparent plant, via tissue culture techniques or a group of inbred plantswhich are genetically very similar due to descent from a commonparent(s). A plant is said to “belong” to a particular line if it (a) isa primary transformant (T0) plant regenerated from material of thatline; (b) has a pedigree comprised of a T0 plant of that line; or (c) isgenetically very similar due to common ancestry (e.g., via inbreeding orselfing). In this context, the term “pedigree” denotes the lineage of aplant, e.g. in terms of the sexual crosses effected such that a gene ora combination of genes, in heterozygous (hemizygous) or homozygouscondition, imparts a desired trait to the plant.

As used herein, the term “locus” (plural: “loci”) refers to any sitethat has been defined genetically. A locus may be a gene, or part of agene, or a DNA sequence that has some regulatory role, and may beoccupied by the same or different sequences.

The invention provides methods for obtaining plants or plant cellsthrough transformation. As used herein, the term “transformation” refersto the transfer of nucleic acid (i.e., a nucleotide polymer) into acell. As used herein, the term “genetic transformation” refers to thetransfer and incorporation of DNA, especially recombinant DNA, into acell.

The invention provides plant and plant cell transformants. As usedherein, the term “transformant” refers to a cell, tissue or organismthat has undergone transformation. The original transformant isdesignated as “T0” or “T₀.” Selfing the T0 produces a first transformedgeneration designated as “T1” or “T₁.”

The invention provides plant transgenes. As used herein, the term“transgene” refers to a nucleic acid that is inserted into an organism,host cell or vector in a manner that ensures its function.

The invention provides plant transgenic plants, plant parts, and plantcells. As used herein, the term “transgenic” refers to cells, cellcultures, organisms (e.g., plants), and progeny which have received aforeign or modified gene by one of the various methods oftransformation, wherein the foreign or modified gene is from the same ordifferent species than the species of the organism receiving the foreignor modified gene.

The invention provides plant transposition events. As used herein, theterm “transposition event” refers to the movement of a transposon from adonor site to a target site.

The invention provides plant varieties. As used herein, the term“variety” refers to a subdivision of a species, consisting of a group ofindividuals within the species that are distinct in form or functionfrom other similar arrays of individuals.

The invention provides plant vectors, plasmids, or constructs. As usedherein, the term “vector”, “plasmid”, or “construct” refers broadly toany plasmid or virus encoding an exogenous nucleic acid. The term shouldalso be construed to include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into virions or cells, such as, forexample, polylysine compounds and the like. The vector may be a viralvector that is suitable as a delivery vehicle for delivery of thenucleic acid, or mutant thereof, to a cell, or the vector may be anon-viral vector which is suitable for the same purpose. Examples ofviral and non-viral vectors for delivery of DNA to cells and tissues arewell known in the art and are described, for example, in Ma et al.(1997. Proc. Natl. Acad. Sci. U.S.A. 94:12744-12746).

The invention provides isolated, chimeric, recombinant or syntheticpolynucleotide sequences. As used herein, the term “polynucleotide”,“polynucleotide sequence”, or “nucleic acid” refers to a polymeric formof nucleotides of any length, either ribonucleotides ordeoxyribonucleotides, or analogs thereof. This term refers to theprimary structure of the molecule, and thus includes double- andsingle-stranded DNA, as well as double- and single-stranded RNA. It alsoincludes modified nucleic acids such as methylated and/or capped nucleicacids, nucleic acids containing modified bases, backbone modifications,and the like. The terms “nucleic acid” and “nucleotide sequence” areused interchangeably. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by a single letterdesignation as follows: “A” for adenylate or deoxyadenylate (for RNA orDNA, respectively), “C” for cytidylate or dcoxycytidylate, “G” forguanylate or dcoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T. “I” for inosine, and “N” forany nucleotide.

The invention provides isolated, chimeric, recombinant or polypeptidesequences. As used herein, the terms “polypeptide,” “peptide,” and“protein” are used interchangeably herein to refer to polymers of aminoacids of any length. These terms also include proteins that arepost-translationally modified through reactions that includeglycosylation, acetylation and phosphorylation.

The invention provides homologous and orthologous polynucleotides andpolypeptides. As used herein, the term “homologous” or “homologue” or“ortholog” is known in the art and refers to related sequences thatshare a common ancestor or family member and are determined based on thedegree of sequence identity. The terms “homology”, “homologous”,“substantially similar” and “corresponding substantially” are usedinterchangeably herein. They refer to nucleic acid fragments whereinchanges in one or more nucleotide bases do not affect the ability of thenucleic acid fragment to mediate gene expression or produce a certainphenotype. These terms also refer to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially alter the functionalproperties of the resulting nucleic acid fragment relative to theinitial, unmodified fragment. It is therefore understood, as thoseskilled in the art will appreciate, that the invention encompasses morethan the specific exemplary sequences. These terms describe therelationship between a gene found in one species, subspecies, variety,cultivar or strain and the corresponding or equivalent gene in anotherspecies, subspecies, variety, cultivar or strain. For purposes of thisinvention homologous sequences are compared. “Homologous sequences” or“homologues” or “orthologs” are thought, believed, or known to befunctionally related. A functional relationship may be indicated in anyone of a number of ways, including, but not limited to: (a) degree ofsequence identity and/or (b) the same or similar biological function.Preferably, both (a) and (b) are indicated. The degree of sequenceidentity may vary, but in one embodiment, is at least 50% (when usingstandard sequence alignment programs known in the art), at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or at least 98.5%, or at least about 99%,or at least 99.5%, or at least 99.8%, or at least 99.9%. Homology can bedetermined using software programs readily available in the art, such asthose discussed in Current Protocols in Molecular Biology (F. M. Ausubelet al., eds., 1987) Supplement 30, section 7.718, Table 7.71. Somealignment programs are MacVector (Oxford Molecular Ltd. Oxford, U.K.),ALIGN Plus (Scientific and Educational Software, Pennsylvania) andAlignX (Vector NTI, Invitrogen, Carlsbad, Calif.). Another alignmentprogram is Sequencher (Gene Codes, Ann Arbor, Mich.), using defaultparameters.

The invention provides polynucleotides with nucleotide change whencompared to a wild-type reference sequence. As used herein, the term“nucleotide change” refers to, e.g., nucleotide substitution, deletion,and/or insertion, as is well understood in the art. For example,mutations contain alterations that produce silent substitutions,additions, or deletions, but do not alter the properties or activitiesof the encoded protein or how the proteins are made.

The invention provides polypeptides with protein modification whencompared to a wild-type reference sequence. As used herein, the term“protein modification” refers to, e.g., amino acid substitution, aminoacid modification, deletion, and/or insertion, as is well understood inthe art.

The invention provides polynucleotides and polypeptides derived fromwild-type reference sequences. As used herein, the term “derived from”refers to the origin or source, and may include naturally occurring,recombinant, unpurified, or purified molecules, and may also includecells whose origin is a plant or plant part. A nucleic acid or an aminoacid derived from an origin or source may have all kinds of nucleotidechanges or protein modification as defined elsewhere herein.

The invention provides portions or fragments of the nucleic acidsequences and polypeptide sequences of the present invention. As usedherein, the term “at least a portion” or “fragment” of a nucleic acid orpolypeptide means a portion having the minimal size characteristics ofsuch sequences, or any larger fragment of the full length molecule, upto and including the full length molecule. For example, a portion of anucleic acid may be 12 nucleotides, 13 nucleotides, 14 nucleotides, 15nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19nucleotides, 20 nucleotides, 22 nucleotides, 24 nucleotides, 26nucleotides, 28 nucleotides, 30 nucleotides, 32 nucleotides, 34nucleotides, 36 nucleotides, 38 nucleotides, 40 nucleotides, 45nucleotides, 50 nucleotides, 55 nucleotides, and so on, going up to thefull length nucleic acid. Similarly, a portion of a polypeptide may be 4amino acids, 5 amino acids, 6 amino acids, 7 amino acids, and so on,going up to the full length polypeptide. The length of the portion to beused will depend on the particular application. A portion of a nucleicacid useful as hybridization probe may be as short as 12 nucleotides; inone embodiment, it is 20 nucleotides. A portion of a polypeptide usefulas an epitope may be as short as 4 amino acids. A portion of apolypeptide that performs the function of the full-length polypeptidewould generally be longer than 4 amino acids.

The invention provides sequences having high similarity or identity tothe nucleic acid sequences and polypeptide sequences of the presentinvention. As used herein, “sequence identity” or “identity” in thecontext of two nucleic acid or polypeptide sequences includes referenceto the residues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17(1988).

The invention provides sequences substantially complementary to thenucleic acid sequences of the present invention. As used herein, theterm “substantially complementary” means that two nucleic acid sequenceshave at least about 65%, preferably about 70% or 75%, more preferablyabout 80% or 85%, even more preferably 90% or 95%, and most preferablyabout 98% or 99%, sequence complementarities to each other. This meansthat primers and probes must exhibit sufficient complementarity to theirtemplate and target nucleic acid, respectively, to hybridize understringent conditions. Therefore, the primer and probe sequences need notreflect the exact complementary sequence of the binding region on thetemplate and degenerate primers can be used. For example, anon-complementary nucleotide fragment may be attached to the 5′-end ofthe primer, with the remainder of the primer sequence beingcomplementary to the strand. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer has sufficient complementarity with the sequence of one of thestrands to be amplified to hybridize therewith, and to thereby form aduplex structure which can be extended by polymerizing means. Thenon-complementary nucleotide sequences of the primers may includerestriction enzyme sites. Appending a restriction enzyme site to theend(s) of the target sequence would be particularly helpful for cloningof the target sequence. A substantially complementary primer sequence isone that has sufficient sequence complementarity to the amplificationtemplate to result in primer binding and second-strand synthesis. Theskilled person is familiar with the requirements of primers to havesufficient sequence complementarity to the amplification template.

The invention provides biologically active variants or functionalvariants of the nucleic acid sequences and polypeptide sequences of thepresent invention. As used herein, the phrase “a biologically activevariant” or “functional variant” with respect to a protein refers to anamino acid sequence that is altered by one or more amino acids withrespect to a reference sequence, while still maintains substantialbiological activity of the reference sequence. The variant can have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. Alternatively, a variant can have “nonconservative” changes,e.g., replacement of a glycine with a tryptophan. Analogous minorvariations can also include amino acid deletion or insertion, or both.Guidance in determining which amino acid residues can be substituted,inserted, or deleted without eliminating biological or immunologicalactivity can be found using computer programs well known in the art, forexample, DNASTAR software. For polynucleotides, a variant comprises apolynucleotide having deletions (i.e., truncations) at the 5′ and/or 3′end; deletion and/or addition of one or more nucleotides at one or moreinternal sites in the reference polynucleotide; and/or substitution ofone or more nucleotides at one or more sites in the referencepolynucleotide. As used herein, a “reference” polynucleotide comprises anucleotide sequence produced by the methods disclosed herein. Variantpolynucleotides also include synthetically derived polynucleotides, suchas those generated, for example, by using site directed mutagenesis butwhich still comprise genetic regulatory element activity. Generally,variants of a particular polynucleotide or nucleic acid molecule of theinvention will have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%,97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, 99.9% or more sequence identity to that particularpolynucleotide as determined by sequence alignment programs andparameters as described elsewhere herein.

Variant polynucleotides also encompass sequences derived from amutagenic and recombinogenic procedure such as DNA shuffling. Strategiesfor such DNA shuffling are known in the art. See, for example, Stemmer(1994) PNAS 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameriet al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.Biol. 272:336-347; Zhang et al. (1997) PNAS 94:4504-4509; Cramcri et al.(1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.For PCR amplifications of the polynucleotides disclosed herein,oligonucleotide primers can be designed for use in PCR reactions toamplify corresponding DNA sequences from eDNA or genomic DNA extractedfrom any plant of interest. Methods for designing PCR primers and PCRcloning are generally known in the art and are disclosed in Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold SpringHarbor Laboratory Press, Plainview, New York). See also Innis et al.,eds. (1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

The invention provides primers that are derived from the nucleic acidsequences and polypcptidc sequences of the present invention. The term“primer” as used herein refers to an oligonucleotide which is capable ofannealing to the amplification target allowing a DNA polymerase toattach, thereby serving as a point of initiation of DNA synthesis whenplaced under conditions in which synthesis of primer extension productis induced, i.e., in the presence of nucleotides and an agent forpolymerization such as DNA polymerase and at a suitable temperature andpH. The (amplification) primer is preferably single stranded for maximumefficiency in amplification. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the agent forpolymerization. The exact lengths of the primers will depend on manyfactors, including temperature and composition (A/T vs. G/C content) ofprimer. A pair of bi-directional primers consists of one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

The invention provides polynucleotide sequences that can hybridize withthe nucleic acid sequences of the present invention. The terms“stringency” or “stringent hybridization conditions” refer tohybridization conditions that affect the stability of hybrids, e.g.,temperature, salt concentration, pH, formamide concentration and thelike. These conditions are empirically optimized to maximize specificbinding and minimize non-specific binding ofprimer or probe to itstarget nucleic acid sequence. The terms as used include reference toconditions under which a probe or primer will hybridize to its targetsequence, to a detectably greater degree than other sequences (e.g. atleast 2-fold over background). Stringent conditions are sequencedependent and will be different in different circumstances. Longersequences hybridize specifically at higher temperatures. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of a complementary target sequence hybridizes to aperfectly matched probe or primer. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M Na⁺ion, typically about 0.01 to 1.0 M Na+ ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes or primers (e.g. 10 to 50 nucleotides) and at least about60° C. for long probes or primers (e.g. greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. Exemplary low stringentconditions or “conditions of reduced stringency” include hybridizationwith a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C. anda wash in 2×SSC at 40° C. Exemplary high stringency conditions includehybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in0.1×SSC at 60° C. Hybridization procedures are well known in the art andare described by e.g. Ausubel et al., 1998 and Sambrook et al., 2001.

The invention provides coding sequences. As used herein, “codingsequence” refers to a DNA sequence that codes for a specific amino acidsequence.

The invention provides regulatory sequences. “Regulatory sequences”refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence.

The invention provides promoter sequences. As used herein, “promoter”refers to a DNA sequence capable of controlling the expression of acoding sequence or functional RNA. The promoter sequence consists ofproximal and more distal upstream elements, the latter elements oftenreferred to as enhancers. Accordingly, an “enhancer” is a DNA sequencethat can stimulate promoter activity, and may be an innate element ofthe promoter or a heterologous element inserted to enhance the level ortissue specificity of a promoter. Promoters may be derived in theirentirety from a native gene, or be composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments. It is understood by those skilled in the artthat different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity.

In some embodiments, the invention provides plant promoters. As usedherein, a “plant promoter” is a promoter capable of initiatingtranscription in plant cells whether or not its origin is a plant cell,e.g. it is well known that Agrobacterium promoters are functional inplant cells. Thus, plant promoters include promoter DNA obtained fromplants, plant viruses and bacteria such as Agrobacterium andBradyrhizohbium bacteria. A plant promoter can be a constitutivepromoter or a non-constitutive promoter.

The invention provides recombinant genes comprising 3′ non-codingsequences or 3′ untranslated regions. As used herein, the “3′ non-codingsequences” or “3′ untranslated regions” refer to DNA sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht, I. L., et al. (1989)Plant Cell 1:671-680.

The invention provides RNA transcripts. As used herein, “RNA transcript”refers to the product resulting from RNA polymerase-catalyzedtranscription of a DNA sequence. When the RNA transcript is a perfectcomplementary copy of the DNA sequence, it is referred to as the primarytranscript. An RNA transcript is referred to as the mature RNA when itis an RNA sequence derived from post-transcriptional processing of theprimary transcript. “Messenger RNA (mRNA)” refers to the RNA that iswithout introns and that can be translated into protein by the cell.“cDNA” refers to a DNA that is complementary to and synthesized from anmRNA template using the enzyme reverse transcriptase. The cDNA can besingle-stranded or converted into the double-stranded form using theKlenow fragment of DNA polymerase 1. “Sense” RNA refers to RNAtranscript that includes the mRNA and can be translated into proteinwithin a cell or in vitro. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA, and that blocks the expression of a target gene (U.S. Pat. No.5,107,065). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns, or the coding sequence. “Functional RNA”refers to antisense RNA, ribozyme RNA, or other RNA that may not betranslated but yet has an effect on cellular processes. The terms“complement” and “reverse complement” are used interchangeably hereinwith respect to mRNA transcripts, and are meant to define the antisenseRNA of the message.

The invention provides recombinant genes in which a gene of interest isoperably linked to a promoter sequence. As used herein, the term“operably linked” refers to the association of nucleic acid sequences ona single nucleic acid fragment so that the function of one is regulatedby the other. For example, a promoter is operably linked with a codingsequence when it is capable of regulating the expression of that codingsequence (i.e., that the coding sequence is under the transcriptionalcontrol of the promoter). Coding sequences can be operably linked toregulatory sequences in a sense or antisense orientation. In anotherexample, the complementary RNA regions of the invention can be operablylinked, either directly or indirectly, 5′ to the target mRNA, or 3′ tothe target mRNA, or within the target mRNA, or a first complementaryregion is 5′ and its complement is 3′ to the target mRNA.

The invention provides recombinant expression cassettes and recombinantconstructs. As used herein, the term “recombinant” refers to anartificial combination of two otherwise separated segments of sequence,e.g., by chemical synthesis or by the manipulation of isolated segmentsof nucleic acids by genetic engineering techniques. As used herein, thephrases “recombinant construct”, “expression construct”, “chimericconstruct”, “construct”, and “recombinant DNA construct” are usedinterchangeably herein. A recombinant construct comprises an artificialcombination of nucleic acid fragments, e.g., regulatory and codingsequences that are not found together in nature. For example, a chimericconstruct may comprise regulatory sequences and coding sequences thatare derived from different sources, or regulatory sequences and codingsequences derived from the same source, but arranged in a mannerdifferent than that found in nature. Such construct may be used byitself or may be used in conjunction with a vector. If a vector is usedthen the choice of vector is dependent upon the method that will be usedto transform host cells as is well known to those skilled in the art.For example, a plasmid vector can be used. The skilled artisan is wellaware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cellscomprising any of the isolated nucleic acid fragments of the invention.The skilled artisan will also recognize that different independenttransformation events will result in different levels and patterns ofexpression (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida et al.,(1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events mustbe screened in order to obtain lines displaying the desired expressionlevel and pattern. Such screening may be accomplished by Southernanalysis of DNA, Northern analysis of mRNA expression, immunoblottinganalysis of protein expression, or phenotypic analysis, among others.Vectors can be plasmids, viruses, bacteriophages, pro-viruses,phagemids, transposons, artificial chromosomes, and the like, thatreplicate autonomously or can integrate into a chromosome of a hostcell. A vector can also be a naked RNA polynucleotide, a naked DNApolynucleotide, a polynucleotide composed of both DNA and RNA within thesame strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugatedDNA or RNA, a liposome-conjugated DNA, or the like, that is notautonomously replicating.

In yet another embodiment, the present invention provides a tissueculture of regenerable cells of a durum wheat plant obtained from thedurum wheat lines of the present invention, wherein the tissueregenerates plants having all or substantially all of the morphologicaland physiological characteristics of the durum wheat plants provided bythe present invention. In one such embodiment, the tissue culture isderived from a plant part selected from the group consisting of leaves,roots, root tips, root hairs, anthers, pistils, stamens, pollen, ovules,flowers, seeds, embryos, stems, buds, cotyledons, hypocotyls, cells andprotoplasts. In another such embodiment, the present invention includesa wheat plant regenerated from the above described tissue culture.

This invention provides the cells, cell culture, tissues, tissueculture, seed, whole plant and plant parts of durum wheat germplasmdesignated ‘DHA175’ or derived from ‘DHA-175’ or any of its offspring.

This invention provides the cells, cell culture, tissues, tissueculture, seed, whole plant and plant parts of durum wheat germplasmdesignated ‘DHA55’ or derived from DHA-55 or any of its offspring. Forexample methods of wheat tissue culture please see (Altpeter et al.,1996; Smidansky et al., 2002)

Wheat

Wheat is a plant species belonging to the genus of Triticum.Non-limiting examples of wheat species include, T. aestivum (a.k.a.,common wheat, or bread wheat, hexaploid), T. aethiopicum, T. araralicum,T. boeoticum, T. carthlicum, T. compactum, T. dicoccoides, T. dicoccum(a.k.a., emmer wheat, tetraploid), T. durum (a.k.a., dunrum wheat,tetraploid). T. ispahanictum, T. karamyschevii. T. macha, T. militinae,T. monococcum (Einkorn wheat, diploid). T. polonicum, T. spella (a.k.a.spelt, hexaploid). T. sphaerococcum, T. limopheevii, T. turanicum, T.lurgidum, T. urartu, T. vavilovii, T. zhukovskyi, and any hybridizationthereof.

Some wheat species are diploid, with two sets of chromosomes, but manyare stable polyploids, with four sets (tetraploid) or six sets(hexaploid) of chromosomes.

Einkorn wheat (T. monococcum) is diploid (AA, two complements of sevenchromosomes, 2n=14). Most tetraploid wheats (e.g. emmer and durum wheat)are derived from wild emmer, T. dicoccoides. Wild emmer is itself theresult of a hybridization between two diploid wild grasses, T. urartuand a wild goatgrass such as Aegilops searsii or Aegilops speltoides.The hybridization that formed wild emmer (AABB) occurred in the wild,long before domestication, and was driven by natural selection (Hancock,James F. (2004) Plant Evolution and the Origin of Crop Species. CABIPublishing. ISBN 0-85199-685-X). Hexaploid wheats (AABBDD) evolved infarmers' fields. Either domesticated emmer or durum wheat hybridizedwith yet another wild diploid grass (Aegilops tauschii) to make thehexaploid wheats, spelt wheat and bread wheat. These have three sets ofpaired chromosomes.

Therefore, in hexaploid wheat, most genes exist in triplicatedhomoeologous sets, one from each genome (i.e., the A genome, the Bgenome, or the D genome), while in tetraploid wheat, most genes exist indoubled homologous sets, one from each genome (i.e., the A genome or theB genome). Due to random mutations that occur along genomes, the allelesisolated from different genomes are not necessarily identical.

The presence of certain alleles of wheat genes is important for cropphenotypes. Some alleles encode functional polypeptides with equal orsubstantially equal activity of a reference allele. Some alleles encodepolypeptides having increased activity when compared to a referenceallele. Some alleles are in disrupted versions which do not encodefunctional polypeptides, or only encode polypeptides having lessactivity compared to a reference allele. Each of the different allelescan be utilized depending on the specific goals of a breeding program.

Wheat Starch Synthesis Genes

Starch is the major reserve carbohydrate in plants. It is present inpractically every type of tissue: leaf, fruit, root, shoot, stem,pollen, and seed. In cereal grains, starch is the primary source ofstored energy. The amount of starch contained in cereal grains variesdepending on species, and developmental stages.

Two types of starch granules are found in the wheat endosperm. The large(A-type) starch granules of wheat are disk-like or lenticular in shape,with an average diameter of 10-35 μm, whereas the small (B-type) starchgranules are roughly spherical or polygonal in shape, ranging from 1 to10 μm in diameter.

Bread wheat (Triticum aestivum L.) starch normally consists of roughly25% amylose and 75% amylopectin (reviewed in Hannah and James, 2008).Amylose is a linear chain of glucose molecules linked by α-1,4 linkages.Amylopectin consists of glucose residues linked by α-1,4 linkages withα-1,6 branch points.

Starch synthesis is catalyzed by starch synthases. Amylose andamylopectin are synthesized by two pathways having a common substrate,ADP-glucose. AGPase catalyzes the initial step in starch synthesis inplants. Waxy proteins granule bound starch synthase I (GBSSI) is encodedby Wx genes which are responsible for amylose synthesis. Soluble starchsynthase, such as starch synthase I (SSI or SI), II (SSII or SII), andIII (SSIII or SIII), starch branching enzymes (e.g., SBEI, SBEIIa andSBEIIb), and starch debranching enzymes of isoamylase- and limitdextrinase-type (ISA and LD) are believed to play key roles inamylopectin synthesis.

SSI of wheat is partitioned between the granule and the soluble fraction(Li et al., 1999, Peng et al., 2001). Wheat SSII is predominantlygranule-bound with only a small amount present in the soluble fraction(Gao and Chibbar, 2000). SSIII is exclusively found in the solublefraction of wheat endosperm (Li et al., 2000).

SBEs can be separated into two major groups. SBE type I (or class B)comprises SBEI from maize (Baba et al, 1991), wheat (Morell et al, 1997,Repellin et al, 1997, Baga et al, 1999b), potato (Kossman et al, 1991),rice (Kawasaki et al, 1993), and cassava (Salehuzzaman et aL, 1992), andSBEII from pea (Burton et aL, 1995). The other group, SBE type II (orclass A), comprises SBEII from maize (Gao et al, 1997), wheat (Nair etal, 1997), potato (Larsson et al, 1996), and Arabidopsis (Fisher et aL,1996), SBEIII from rice (Mizuno et al, 1993), and SBEI from pea(Bhattacharyya et al, 1990). SBEI and SBEII are generallyimmunologically unrelated but have distinct catalytic activities. SBEItransfers long glucan chains and prefers amylose as a substrate, whileSBEII acts primarily on amylopectin (Guan and Preiss, 1993). SBEII isfurther subclassified into SBElla and SBEllb, each of which differsslightly in catalytic properties. The two SBEII forms are encoded bydifferent genes and expressed in a tissue-specific manner (Gao et al.,1997, Fisher et al., 1996). Expression patterns of SBElla and SBEllb ina particular tissue are specific to plant species. For example, theendosperm-specific SBEII in rice is SBElla (Yamanouchi and Nakamura,1992), while that in barley is SBEllb (Sun et al., 1998).

SDBE can be either alpha-1,4-targeting enzymes, such as amylases, starchphosphorylase (EC 2.4.1.1), disproportionating enzyme (EC 2.4.1.25), oralpha-1,6-targeting enzymes, such as direct debranching enzymes (e.g.,limit dextrinase, EC 3.2.1.41, or isoamylase. EC 3.2.2.68), indirectdebranching enzymes (e.g., alpha-1,4- and alpha-4,6-targeting enzymes).

Several starch biosynthetic proteins can be found bound to the interiorof starch granules. A subset of these proteins has been designated thestarch granule proteins (SGPs). Bread wheat starch granule proteins(SGPs) at least include SGP-1, SGP-2 and SGP-3 all with molecularmasses >80 kd and the waxy protein (GBSS). The SGP-1 fraction of breadwheat was resolved into SGP-A1, SGP-B1, and SGP-D1, and genes encodingthese proteins were localized to homoeologous group 7 chromosomes(Yamamori and Endo, 1996). Increased Amylose is observed by about 8% inthe SGP-1 null line compared to the wild type inferring that SGP-1 isinvolved in amylopectin synthesis (Yamamori et al. (2000). The SGP-1null line also shows deformed starch granules, lower overall starchcontent, altered amylopectin content, and reduced binding of SGP-2 andSGP-3 to starch granules. SGP-1 proteins are starch synthase class IIenzymes and genes encoding these enzymes are designated SSII-A1,SSII-B1, and SSII-D1 (Li et al., 1999).

Durum wheat (Triticum lurgidum L. var. durum) being tetraploid lacks theD genome of bread wheat but homoalleles for genes encoding the SGP-1proteins are present on the A and B genomes (Lafiandra et al., 2010).The hexaploid SGP-A1 and SGP-B1 mutants from Yamamori and Endo (1996)were crossed into to durum cultivar Svevo. The SGP-A1/B1 null progenyexhibited 20% higher amylose content than Svevo wild type wheat, and hadreduced binding of SGP-2 and SGP-3 to starch granules. These crossesbetween hexaploid bread wheat and tetraploid durum however are notconsidered commercially viable products.

Progeny of durum×hexaploid crosses are highly variable due to thevariable incorporation of A and B genome loci with parental choicehaving a large impact upon cross success rates (Lanning et al. 2008;Martin et al. 2011). Moreover, the agronomic yield of lines fromtetraploid×hexaploid wheat crosses would be expected to be lower thanthe adapted parents due to break up of adapted gene complexes. Thedisadvantages of hexaploid×durum crosses are well known in the art andto the present inventor's knowledge, no commonly grown durum varietieshave resulted from crosses between durum and hexaploid wheat varieties.Therefore, the creation of high amylose durum wheat by specificallyselecting for mutations in the durum starch synthase 11 genes ispreferable to integration of hexaploid wheat starch synthase 11mutations by crossing with durum wheat.

SGP-1 mutations are thought to alter the interactions of other granulebound enzymes by reducing their entrapment in starch granules.Similarly, barley SSIIa sex6 locus mutations have seeds with decreasedstarch content, increased amylose content (+45%) (70.3% for two SGP-1mutants vs. 25.4% wild-type), deformed starch granules, and decreasedbinding of other SGPs (Morell et al. 2003). These barley ssIIa mutantshad normal expression of SSI, SBEIIa, and SBEIIb based on western blotanalysis of the soluble protein fraction demonstrating that there wasnot a global down regulation of starch synthesis genes. In SGP-1 triplemutant in bread wheat, SSI, SBEIIa, and SBEIIb proteins were stablyexpressed in developing seeds even though they are not present in thestarch granule fraction (Kosar-Hashemi et al. 2007). Similar resultsrelating the loss of SSII and increased amylose have been observed inboth maize (Zhang et al. 2004) and pea (Craig et al. 1998).

Elimination of another important gene for amylopectin synthesis, Sbella,in durum wheat through RNA interference resulted in amylose increasesranging from +8% to +50% (24% wild-type vs. 31-75% SbeIIa RNAi lines),although protein content was found to be similar or, in some cases,lower than wild type. (Sestili et al. 2010b). It was determined throughqRT-PCR that the silencing of SbeIIa resulted in elevated expression ofthe Waxy genes, SSIII, limit dextrinase (Ld1), and isoamylase-1 (Iso1).The very high amylose results observed by Sestili et al. (2010b) in someof their transgenic lines may not have been due solely to reduction ofSbeIIa expression since SbeIIa mutagenesis resulted in amylose levelsincreases more similar to those of SSIIa mutations (28% sbeIIa doublemutant versus 23% wild-type) (Hazard et al. 2012). To date a detailedexpression profile of starch synthesis genes in a SGP-1 null backgroundhas not been reported. RNA-Seq is an emerging method that employsnext-generation sequencing technologies that allow for gene expressionanalysis at the transcript level. RNA-Seq offers single-nucleotideresolution that is highly reproducible (Marioni et al. 2008) andcompared to other methods has a greater sequencing sensitivity, a largedynamic range, and the ability to distinguish between differing allelesor isoforms of an expressed gene. RNA-Scq is therefore an ideal methodto use to determine the effect a null SGP-1 genotype has on expressionof other starch synthesis genes.

Cereals with high amylose content are desirable because they have moreresistant starch. Resistant starch is starch that resists break down inthe intestines of humans and animals and thus acts more like dietaryfiber while promoting microbial fermentation (reviewed in Nugent 2005).Products that have high resistant starch levels are viewed as healthy asthey increase overall colon health and decrease sugar release duringfood digestion. Rats fed whole seed meal from SbeIIa RNAi silenced breadwheat with an amylose content of 80% showed significant improvements inbowel health indices and increases in short-chained fatty acids (SCFAs),the end products of microbial fermentation (Regina et al. 2006).Similarly, when null ssIIa barley was fed to humans there wassignificant improvement in several bowel health indices and increases inSCFAs (Bird et al. 2008). An extruded cereal made from the ssIIa nullbarley also resulted in a lower glycemic index and lower plasma insulinresponse when fed to humans (King et al. 2008). The Yamamori et al.(2000) SGP-1 single mutants were crossed and backcrossed to an Italianbreeding line then interbred to produce a triple null line from whichwhole grain bread was prepared. The resultant bread with the addition oflactic acid had increased resistant starch and a decreased glycemicindex, but did not impact insulin levels (Hallstrom et al. 2011).Recently a high amylose corn was shown to alter insulin sensitivity inoverweight men making them less likely to have insulin resistance, thepathophysiologic feature of diabetes (Maki et al. 2012).

In addition to the positive impact of increased amylose upon glycemicindex, higher amylose could result in enhanced durum product quality.Pasta that is firmer when cooked is preferred as it resists overcookingand it is expected that high amylose should result in increased noodlefirmness. Resistance to overcooking is positively correlated with pastafirmness. Current high amylose wheat based foods are prepared usingstandard amylose content wheat flour with the addition of high amylosemaize starch (Thompson, 2000). To test the impact of high amylose upondurum quality Soh et al. (2006) varied durum flour amylose content byreconstituting durumn flour with the addition of high amylose maizestarch and wheat gluten. The increased amylose flours had weaker lessextensible dough but resulted in firmer pasta. Pastas are a popular fooditem globally and are primarily made from durum semolina which is alsoutilized in a host of other culturally important foods. In someembodiments, the present invention develops a high-amylose durum linethrough the creation of mutations in SSIIa and to examine the effect aSGP-1 null genotype has on the expression of other genes involved instarch synthesis using RNA-Seq. These lines are tested for their endproduct quality and potential health benefits.

The ratio of amylose to amylopectin can be changed by selecting foralternate forms of the Wx loci or other starch synthase loci. Breadwheats carrying the null allele at all three Wx loci (Nakamura, et al.,1995) and durum wheat (Lafiandra et al., 2010 and Vignaux et at., 2004)with null alleles at both Wx loci are nearly devoid of amylose. On theother hand, bread wheat lines null at the three SGP-1 loci had 37.5%amylose compared to 24.9% amylose for the wild type genotype, determinedby differential scanning calorimetry (Morita et al., 2005). Durum wheatlines with null alleles for both SGP-1 loci had 43.6% amylose comparedto 23.0% for the wild type genotype (Lafiandra et al., 2010). Genotypeswith a null allele at only one of the Wx loci (partial waxy) show onlysmall reductions in amylose content. For example, Martin et al. (2004)showed a 2.4% difference in amylose between the wild type and nullalleles in a recombinant inbred population segregating for Wx-B1.Vignaux et at., (2004) showed partial waxy durum genotypes reducedamylose by 1% but that difference was not significant.

High Fiber and Amylose Flour and Resulting Products

In Europe and in North America, pasta is traditionally prepared using100% durum flour (Fuad and Prabhasanker 2010). In fact, the propertiesinherent in durum wheat flour make it ideally suited for pastaproduction since it imparts excellent color due to relatively highyellow pigments levels and good mixing properties inherent in nativeglutenin proteins (Dexter and Matson 1979; Fuad and Prabhasanker 2010).Recently, there has been a movement towards the production of flourproducts with improved nutritional properties including increased fiberand amylose content, as well as flour products having increased proteincontent.

Flour with increased dietary fiber is associated with bettergastrointestinal health, and lower risk of diabetes and heart disease.Flour with high amylose content is also desirable as it has a highercontent of resistant starch that is not absorbed during digestion andthus produces health benefits similar to those of dietary fiber. Theincreased amylose content of flour also influences the gelatinizationand pasting properties of starch. Peak viscosity, final viscosity, breakdown, set back and peak time measured by Rapid Visco Analyzer (RVA) alldeclined with increasing amylose content for durum wheat (Lafiandra etal., 2010). The altered starch properties translate into changes in endproduct properties such as increased firmness and resistance toovercooking.

Increasing the dietary fiber, amylose, and/or protein content of wheatflour products can be achieved by incorporating various protein ordietary fiber enriched fractions such as pea flour, cereal-soluble orinsoluble fiber. These types of mixed enriched flour blends however canlead to consumer acceptance issues. For example, blending barley flourinto durum wheat to increase dietary fiber in pasta led to a darkcolored product (Casiraghi et al., 2013). Fortification of pasta withpea flour deteriorated dough handling characteristics, and increasedpasta cooking losses and led to lower tolerance to overcooking (Nielsenet al., 1980). Modifying durum wheat to increase amylose, protein, anddietary fiber is preferable to durum flour additives since it wouldresult in a pasta having the improved nutrition while also retainingmany of the desirable properties of durum flour. The final product thenwould match the North American and European preference for 100% durumpasta. Durum wheat flour with increased amylose, protein, and dietaryfiber used in the preparation of pasta would likely be preferable evento that of standard whole grain durum pasta which is much darker inappearance and has reduced cooked firmness leading to reduced consumeracceptability (Manthey and Schorno 2002).

There has been recent interest in flours with higher amylose for foodproducts. The main reason being that starch high in amylose has a higherfraction of resistant starch. Resistant starch is that fraction notabsorbed in the small intestine during digestion (reviewed in Nugent2005). Resistant starch is believed to provide health benefits similarto dietary fiber. Commercial high amylose food products havetraditionally been developed using high amylose maize starch (Thompson,2000). The development of high amylose bread wheat genotypes has made itpossible to test the impact of high amylose wheat starch on end productquality. High amylose wheat flour produced harder textured dough andmore viscous, and bread loaves that were smaller than normal flour(Morita et al., 2002). Substituting up to 50% high amylose wheat flourwith the remainder being normal wheat flour gave bread quality that wasnot significantly different from the 100% normal wheat flour control(Hung et al., 2005). Durum wheat flours varying in amylose content canbe made by reconstituting them with high amylose maize starch (Soh etal., 2006). The high amylose durum wheat flours had dough that wasweaker and less extensible. The pasta produced from these flours tendedto be firmer with more cooking loss with increasing amylose content.

Even small, incremental increases in amylose may impact end productquality. Consumers prefer pasta that is firm and is tolerant to overcooking. Reduced amylose produces noodles that are softer in texture(Oda et al 1980; Miura and Tanii 1994; Zhao et al 1998). The impact ofsmall increases in amylose content on durum product quality is notknown. For example, attention has been devoted to Asian noodle qualityfrom partial waxy flours. Partial waxy soft wheat cultivars, due to amutation at one of the Wx loci, are preferred for udon noodles as theyconfer softer texture to the noodles (Oda et al 1980; Miura and Tanii1994; Zhao et al 1998). Partial waxy genotype did not differ from wildtype for white salted noodle firmness in a hard wheat recombinant inbredpopulation (Martin et al., 2004). However, partial waxy genotypeconferred greater loaf volume and bread was softer textured than thatfrom the wild type.

Waxy durum isolines produced pasta that was softer with more cookingloss and which was less resistant to over cooking than pasta from normallines. However, the partial waxy isolines produced pasta with propertiesnot statistically different from the wild type lines (Vignaux et al.,2005).

The present inventors surveyed world durum wheat germplasm andidentified two genotypes that lacked the SGP-A1 protein. These genotypeswere crossed to an adapted durum genotype to create populationssegregating for the SSIIa-Ab null allele. Influence of allelic variationat the SSII-A1 locus on semolina properties and end product qualityusing noodles as a test product were investigated.

Identification and Creation of Mutant Starch Synthesis Genes in Durum

Durum wheat with one or more mutant alleles of one or more starchsynthesis genes can be created and identified. In some embodiments, suchmutant alleles happen naturally during evolution. In some embodiments,such mutant alleles are created by artificial methods, such asmutagenesis (e.g., chemical mutagenesis, radiation mutagenesis,transposon mutagenesis, insertional mutagenesis, signature taggedmutagenesis, site-directed mutagenesis, and natural mutagenesis),antisense, knock-outs, and/or RNA interference.

Various types of mutagenesis can be used to produce and/or isolatevariant nucleic acids that encode for protein molecules and/or tofurther modify/mutate the proteins of a starch synthesis gene. Theyinclude but are not limited to site-directed, random point mutagenesis,homologous recombination (DNA shuffling), mutagenesis using uracilcontaining templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like. Additional suitable methods include pointmismatch repair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis, double-strand breakrepair, and the like. Mutagenesis, e.g., involving chimeric constructs,is also included in the present invention. In one embodiment,mutagenesis can be guided by known information of the naturallyoccurring molecule or altered or mutated naturally occurring molecule,e.g., sequence, sequence comparisons, physical properties, crystalstructure or the like. For more information of mutagenesis in plants,such as agents, protocols, sec Acquaah et al. (Principles of plantgenetics and breeding, Wiley-Blackwell, 2007, ISBN 1405136464,9781405136464, which is herein incorporated by reference in its entity).Methods of disrupting plant genes using RNA interference is describedlater in the specification.

Gene function can also be interrupted and/or altered by RNA interference(RNAi). RNAi is the process of sequence-specific, post-transcriptionalgene silencing or transcriptional gene silencing in animals and plants,initiated by double-stranded RNA (dsRNA) that is homologous in sequenceto the silenced gene. The preferred RNA effector molecules useful inthis invention must be sufficiently distinct in sequence from any hostpolynucleotide sequences for which function is intended to beundisturbed after any of the methods of this invention are performed.Computer algorithms may be used to define the essential lack of homologybetween the RNA molecule polynucleotide sequence and host, essential,normal sequences.

The term “dsRNA” or “dsRNA molecule” or “double-strand RNA effectormolecule” refers to an at least partially double-strand ribonucleic acidmolecule containing a region of at least about 19 or more nucleotidesthat are in a double-strand conformation. The double-stranded RNAeffector molecule may be a duplex double-stranded RNA formed from twoseparate RNA strands or it may be a single RNA strand with regions ofself-complementarity capable of assuming an at least partiallydouble-stranded hairpin conformation (i.e., a hairpin dsRNA or stem-loopdsRNA). In various embodiments, the dsRNA consists entirely ofribonucleotides or consists of a mixture of ribonucleotides anddeoxynucleotides, such as RNA/DNA hybrids. The dsRNA may be a singlemolecule with regions of self-complementarity such that nucleotides inone segment of the molecule base pair with nucleotides in anothersegment of the molecule. In one aspect, the regions ofself-complementarity are linked by a region of at least about 3-4nucleotides, or about 5, 6, 7, 9 to 15 nucleotides or more, which lackscomplementarity to another part of the molecule and thus remainssingle-stranded (i.e., the “loop region”). Such a molecule will assume apartially double-stranded stem-loop structure, optionally, with shortsingle stranded 5′ and/or 3′ ends. In one aspect the regions ofself-complementarity of the hairpin dsRNA or the double-stranded regionof a duplex dsRNA will comprise an Effector Sequence and an EffectorComplement (e.g., linked by a single-stranded loop region in a hairpindsRNA). The Effector Sequence or Effector Strand is that strand of thedouble-stranded region or duplex which is incorporated in or associateswith RISC. In one aspect the double-stranded RNA effector molecule willcomprise an at least 19 contiguous nucleotide effector sequence,preferably 19 to 29, 19 to 27, or 19 to 21 nucleotides, which is areverse complement to a starch synthesis gene.

In some embodiments, the dsRNA effector molecule of the invention is a“hairpin dsRNA”, a “dsRNA hairpin”, “short-hairpin RNA” or “shRNA”,i.e., an RNA molecule of less than approximately 400 to 500 nucleotides(nt), or less than 100 to 200 nt, in which at least one stretch of atleast 15 to 100 nucleotides (e.g., 17 to 50 nt, 19 to 29 nt) is basedpaired with a complementary sequence located on the same RNA molecule(single RNA strand), and where said sequence and complementary sequenceare separated by an unpaired region of at least about 4 to 7 nucleotides(or about 9 to about 15 nt, about 15 to about 100 nt, about 100 to about1000 nt) which forms a single-stranded loop above the stem structurecreated by the two regions of base complementarity. The shRNA moleculescomprise at least one stem-loop structure comprising a double-strandedstem region of about 17 to about 500 bp; about 17 to about 50 bp; about40 to about 100 bp; about 18 to about 40 bp; or from about 19 to about29 bp; homologous and complementary to a target sequence to beinhibited; and an unpaired loop region of at least about 4 to 7nucleotides, or about 9 to about 15 nucleotides, about 15 to about 100nt, about 250-500 bp, about 100 to about 1000 nt, which forms asingle-stranded loop above the stem structure created by the two regionsof base complementarity. It will be recognized, however, that it is notstrictly necessary to include a “loop region” or “loop sequence” becausean RNA molecule comprising a sequence followed immediately by itsreverse complement will tend to assume a stem-loop conformation evenwhen not separated by an irrelevant “stuffer” sequence.

The expression construct of the present invention comprising DNAsequence which can be transcribed into one or more double-stranded RNAeffector molecules can be transformed into a wheat plant, wherein thetransformed plant produces different starch compositions than theuntransformed plant. The target sequence to be inhibited by the dsRNAeffector molecule include, but are not limited to, coding region, 5′ UTRregion, 3′ UTR region of fatty acids synthesis genes.

The effects of RNAi can be both systemic and heritable in plants. Inplants, RNAi is thought to propagate by the transfer of siRNAs betweencells through plasmodesmata. The heritability comes from methylation ofpromoters targeted by RNAi; the new methylation pattern is copied ineach new generation of the cell. A broad general distinction betweenplants and animals lies in the targeting of endogenously producedmiRNAs; in plants, miRNAs are usually perfectly or nearly perfectlycomplementary to their target genes and induce direct mRNA cleavage byRISC, while animals' miRNAs tend to be more divergent in sequence andinduce translational repression. Detailed methods for RNAi in plants aredescribed in David Allis et al (Epigenetics, CSHL Press, 2007, ISBN0879697245, 9780879697242), Sohail et al (Gene silencing by RNAinterference: technology and application, CRC Press, 2005, ISBN0849321417, 9780849321412), Engelke et al. (RAN Interference, AcademicPress, 2005, ISBN 0121827976, 9780121827977), and Doran et al. (RNAInterference: Methods for Plants and Animals, CABI, 2009, ISBN1845934105, 9781845934101), which are all herein incorporated byreference in their entireties for all purposes.

In some embodiments, mutant starch synthesis genes in durum wheat can beidentified by screening durum wheat populations based on one or morephenotypes. In some embodiments, the phenotype is changes in flourswelling power.

In some embodiments, mutant starch synthesis genes in durum wheat can beidentified by screening durum wheat populations based on PCTamplification and sequencing of one or more starch synthesis genes indurum wheat.

In some embodiments, mutant starch synthesis genes in durum wheat can beidentified by TILLING®. Detailed description on methods and compositionson TILLING® can be found in U.S. Pat. No. 5,994,075, US 2004/0053236 A1,WO 2005/055704, and WO 2005/048692, each of which is hereby incorporatedby reference for all purposes.

TILLING® (Targeting Induced Local Lesions in Genomes) is a method inmolecular biology that allows directed identification of mutations in aspecific gene. TILLING® was introduced in 2000, using the model plantArabidopsis thaliana. TILLING® has since been used as a reverse geneticsmethod in other organisms such as zebrafish, corn, wheat, rice, soybean,tomato and lettuce. The method combines a standard and efficienttechnique of mutagenesis with a chemical mutagen (e.g., Ethylmethanesulfonate (EMS)) with a sensitive DNA screening-technique thatidentifies single base mutations (also called point mutations) in atarget gene. EcoTILLING is a method that uses TILLING® techniques tolook for natural mutations in individuals, usually for populationgenetics analysis. See Comai, et al., 2003, Efficient discovery of DNApolymorphisms in natural populations by EcoTILLING. The Plant Journal37, 778-786. Gilchrist et al. 2006. Use of EcoTILLING as an efficientSNP discovery tool to survey genetic variation in wild populations ofPopulus trichocarpa. Mol. Ecol. 15, 1367-1378. Mejlhede et al. 2006.EcoTILLING for the identification of allelic variation within thepowdery mildew resistance genes mlo and Mla of barley. Plant Breeding125, 461-467. Nieto et al. 2007, EcoTILLING for the identification ofallelic variants of melon eIF4E, a factor that controls virussusceptibility. BMC Plant Biology 7, 34-42, each of which isincorporated by reference hereby for all purposes. DEcoTILLING is amodification of TILLING® and EcoTILLING which uses an inexpensive methodto identify fragments (Garvin et al., 2007, DEco-TILLING: An inexpensivemethod for SNP discovery that reduces ascertainment bias. MolecularEcology Notes 7, 735-746).

The invention also encompasses mutants of a starch synthesis gene. Insome embodiments, the starch synthesis gene is selected from the groupconsisting of genes encoding GBSS, waxy proteins, SBE I and II, starchdc-branching enzymes, and SSI, SSII, SSIII, and SSIV. In someembodiments, the starch synthesis gene is SSII. The mutant may containalterations in the amino acid sequences of the constituent proteins. Theterm “mutant” with respect to a polypeptide refers to an amino acidsequence that is altered by one or more amino acids with respect to areference sequence. The mutant can have “conservative” changes, or“nonconservative” changes, e.g., analogous minor variations can alsoinclude amino acid deletions or insertions, or both.

The mutations in a starch synthesis gene can be in the coding region orthe non-coding region of the starch synthesis genes. The mutations caneither lead to, or not lead to amino acid changes in the encoded starchsynthesis gene. In some embodiments, the mutations can be missense,severe missense, silent, nonsense mutations. For example, the mutationcan be nucleotide substitution, insertion, deletion, or genomere-arrangement, which in turn may lead to reading frame shift, aminoacid substitution, insertion, deletion, and/or polypeptides truncation.As a result, the mutant starch synthesis gene encodes a starch synthesispolypeptide having modified activity on compared to a polypeptideencoded by a reference allele.

As used herein, a nonsense mutation is a point mutation, e.g., asingle-nucleotide polymorphism (SNP), in a sequence of DNA that resultsin a premature stop codon, or a nonsense codon in the transcribed mRNA,and in a truncated, incomplete, and usually nonfunctional proteinproduct. A missense mutation (a type of nonsynonymous mutation) is apoint mutation in which a single nucleotide is changed, resulting in acodon that codes for a different amino acid (mutations that change anamino acid to a stop codon are considered nonsense mutations, ratherthan missense mutations). This can render the resulting proteinnonfunctional. Silent mutations are DNA mutations that do not result ina change to the amino acid sequence of a protein. They may occur in anon-coding region (outside of a gene or within an intron), or they mayoccur within an exon in a manner that does not alter the final aminoacid sequence. A severe missense mutation changes the amino acid, whichlead to dramatic changes in conformation, charge status etc.

The mutations can be located at any portion of a starch synthesis gene,for example, at the 5′, the middle, or the 3′ of a starch synthesisgene, resulting mutations in any potions of the encoded starch synthesisprotein.

Mutant starch synthesis protein of the present invention can have one ormore modifications to the reference allele, or biologically activevariant, or fragment thereof. Particularly suitable modificationsinclude amino acid substitutions, insertions, deletions, or truncation.In some embodiments, at least one non-conservative amino acidsubstitution, insertion, or deletion in the protein is made to disruptor modify the protein activity. The substitutions may be single, whereonly one amino acid in the molecule has been substituted, or they may bemultiple, where two or more amino acids have been substituted in thesame molecule. Insertional mutants are those with one or more aminoacids inserted immediately adjacent to an amino acid at a particularposition in the reference protein molecule, biologically active variant,or fragment thereof. The insertion can be one or more amino acids. Theinsertion can consist, e.g., of one or two conservative amino acids.Amino acids similar in charge and/or structure to the amino acidsadjacent to the site of insertion are defined as conservative.Alternatively, mutant starch synthesis protein includes the insertion ofan amino acid with a charge and/or structure that is substantiallydifferent from the amino acids adjacent to the site of insertion. Insome other embodiments, the mutant starch synthesis protein is atruncated protein losing one or more domains compared to a referenceprotein.

In some examples, mutants can have at least 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 40, 50, or 100 amino acid changes. In some embodiments, at leastone amino acid change is a conserved substitution. In some embodiments,at least one amino acid change is a non-conserved substitution. In someembodiments, the mutant protein has a modified enzymatic activity whencompared to a wild type allele. In some embodiments, the mutant proteinhas a decreased or increased enzymatic activity when compared to a wildtype allele. In some embodiments, the decreased or increased enzymaticactivity when compared to a wild type allele leads to amylose contentchange in the durum wheat.

Conservative amino acid substitutions are those substitutions that, whenmade, least interfere with the properties of the original protein, thatis, the structure and especially the function of the protein isconserved and not significantly changed by such substitutions.Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain. Furtherinformation about conservative substitutions can be found, for instance,in Ben Bassat et al. (J. Bacteriol., 169:751-757, 1987), O'Regan et al.(Gene, 77:237-251, 1989), Sahin-Toth et al. (Protein Sci., 3:240-247,1994), Hochuli et al. (Bio/Technology, 6:1321-1325, 1988) and in widelyused textbooks of genetics and molecular biology. The Blosum matricesare commonly used for determining the relatedness of polypeptidesequences. The Blosum matrices were created using a large database oftrusted alignments (the BLOCKS database), in which pairwise sequencealignments related by less than some threshold percentage identity werecounted (Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919,1992). A threshold of 90% identity was used for the highly conservedtarget frequencies of the BLOSUM90 matrix. A threshold of 65% identitywas used for the BLOSUM65 matrix. Scores of zero and above in the Blosummatrices are considered “conservative substitutions” at the percentageidentity selected. The following table shows exemplary conservativeamino acid substitutions.

Very Highly- Highly Conserved Original Conserved Substitutions (from theConserved Substitutions Residue Substitutions Blosum90 Matrix) (from theBlosum65 Matrix) Ala Ser Gly, Ser, Thr Cys, Gly, Ser, Thr, Val Arg LysGln, His, Lys Asn, Gln, Glu, His, Lys Asn Gln; His Asp, Gln, His, Lys,Ser, Thr Arg, Asp, Gln, Glu, His, Lys, Ser, Thr Asp Glu Asn, Glu Asn,Gln, Glu, Ser Cys Ser None Ala Gln Asn Arg, Asn, Glu, His, Lys, Met Arg,Asn, Asp, Glu, His, Lys, Met, Ser Glu Asp Asp, Gln, Lys Arg, Asn, Asp,Gln, His, Lys, Ser Gly Pro Ala Ala, Ser His Asn; Gln Arg, Asn, Gln, TyrArg, Asn, Gln, Glu, Tyr Ile Leu; Val Leu, Met, Val Leu, Met, Phe, ValLeu Ile; Val Ile, Met, Phe, Val Ile, Met, Phe, Val Lys Arg; Gln; GluArg, Asn, Gln, Glu Arg, Asn, Gln, Glu, Ser, Met Leu; Ile Gln, Ile, Leu,Val Gln, Ile, Leu, Phe, Val Phe Met; Leu; Tyr Leu, Trp, Tyr Ile, Leu,Met, Trp, Tyr Ser Thr Ala, Asn, Thr Ala, Asn, Asp, Gln, Glu, Gly, Lys,Thr Thr Ser Ala, Asn, Ser Ala, Asn, Ser, Val Trp Tyr Phe, Tyr Phe, TyrTyr Trp; Phe His, Phe, Trp His, Phe, Trp Val Ile; Leu Ile, Leu, Met Ala,Ile, Leu, Met, Thr

In some embodiments, the mutant durum wheat comprises mutationsassociated with a starch synthesis gene of the same genome that can betraced back to one common ancestor, such as the “A” type genome of durumwheat or the “B” type genome of durum wheat. For example, a mutant durumwheat having a mutated SSIIa-A or a mutated SSIIa-B is included. In someembodiments, one or both alleles of the starch synthesis gene within agiven type of genome are mutated.

In some embodiments, the mutant durum wheat comprise mutationsassociated with the same starch synthesis gene of different genomes thatcan be traced back to two common ancestors, such as the “A” type genomeand the “B” type genome of durum wheat. For example, a mutant durumwheat having a mutated SSIIa-A and a mutated SSIIa-B is included. Insome embodiments, one or both alleles of the starch synthesis genewithin the two types of genomes are mutated.

Methods of Modifying Durum Phenotypes

The present invention further provides methods ofmodifying/altering/improving durum phenotypes. As used herein, the term“modifying” or “altering” refers to any change of phenotypes whencompared to a reference variety, e.g., changes associated with starchproperties. The term “improving” refers to any change that makes thedurum wheat better in one or more qualities for industrial ornutritional applications. Such improvement includes, but is not limitedto, improved quality as meal, improved quality as raw material toproduce a wide range of end products.

In some embodiments, the modified/altered/improved phenotypes arerelated to starch. Starch is the most common carbohydrate in the humandiet and is contained in many foods. The major sources of starch intakeworldwide are the cereals (rice, wheat, and maize) and the rootvegetables (potatoes and cassava). Widely used prepared foods containingstarch are bread, pancakes, cereals, noodles, pasta, porridge andtortilla. The starch industry extracts and refines starches from seeds,roots and tubers, by wet grinding, washing, sieving and drying. Today,the main commercial refined starches are cornstarch, tapioca, wheat andpotato starch.

Starch can be hydrolyzed into simpler carbohydrates by acids, variousenzymes, or a combination of the two. The resulting fragments are knownas dextrins. The extent of conversion is typically quantified bydextrose equivalent (DE), which is roughly the fraction of theglycosidic bonds in starch that have been broken.

Some starch sugars are by far the most common starch based foodingredient and are used as sweetener in many drinks and foods. Theyinclude, but are not limited to, maltodextrin, various glucose syrup,dextrose, high fructose syrup, and sugar alcohols.

A modified starch is a starch that has been chemically modified to allowthe starch to function properly under conditions frequently encounteredduring processing or storage, such as high heat, high shear, low pH,freeze/thaw and cooling. Typical modified starches for technicalapplications are cationic starches, hydroxyethyl starch andcarboxymethylated starches.

As an additive for food processing, food starches are typically used asthickeners and stabilizers in foods such as puddings, custards, soups,sauces, gravies, pie fillings, and salad dressings, and to make noodlesand pastas.

In the pharmaceutical industry, starch is also used as an excipient, astablet disintegrant or as binder.

Starch can also be used for industrial applications, such aspapermaking, corrugated board adhesives, clothing starch, constructionindustry, manufacture of various adhesives or glues for book-binding,wallpaper adhesives, paper sack production, tube winding, gummed paper,envelope adhesives, school glues and bottle labeling. Starchderivatives, such as yellow dextrins, can be modified by addition ofsome chemicals to form a hard glue for paper work; some of those formsuse borax or soda ash, which are mixed with the starch solution at50-70° C. to create a very good adhesive.

Starch is also used to make some packing peanuts, and some drop ceilingtiles. Textile chemicals from starch are used to reduce breaking ofyarns during weaving; the warp yarns are sized. Starch is mainly used tosize cotton based yarns. Modified starch is also used as textileprinting thickener. In the printing industry, food grade starch is usedin the manufacture of anti-set-off spray powder used to separate printedsheets of paper to avoid wet ink being set off. Starch is used toproduce various bioplastics, synthetic polymers that are biodegradable.An example is polylactic acid. For body powder, powdered starch is usedas a substitute for talcum powder, and similarly in other health andbeauty products. In oil exploration, starch is used to adjust theviscosity of drilling fluid, which is used to lubricate the drill headand suspend the grinding residue in petroleum extraction. Glucose fromstarch can be further fermented to biofuel corn ethanol using the socalled wet milling process. Today most bioethanol production plants usethe dry milling process to ferment corn or other feedstock directly toethanol. Hydrogen production can use starch as the raw material, usingenzymes.

Resistant starch is starch that escapes digestion in the small intestineof healthy individuals. High amylose starch from corn has a highergelatinization temperature than other types of starch and retains itsresistant starch content through baking, mild extrusion and other foodprocessing techniques. It is used as an insoluble dietary fiber inprocessed foods such as bread, pasta, cookies, crackers, pretzels andother low moisture foods. It is also utilized as a dietary supplementfor its health benefits. Published studies have shown that Type 2resistant corn helps to improve insulin sensitivity, increases satietyand improves markers of colonic function. It has been suggested thatresistant starch contributes to the health benefits of intact wholegrains.

Resistant starch can be produced from the durum wheat plants of thepresent invention. The resistant starch may have one or more thefollowing features:

-   -   Fiber fortification: the resistant starch is good or excellent        fiber source. The United States Department of Agriculture and        the health organizations of other foreign countries set the        standards for what constitutes a good or excellent source of        dietary fiber.    -   Low caloric contribution: the starch may contain less than about        10 kcal/g, 5 kcal/g, 1 kcal/g, or 0.5 kcal/g, which results in        about 90% calorie reduction compared to typical starch.    -   Low glycemic/insulin response    -   Good flour replacement, because it is (1) easy to be        incorporated into formulations with minimum or no formulation        changes necessary, (2) natural fit for wheat-based products,        and (3) potential to reduce retrogradation and staling. Staling        is a chemical and physical process in bread and other foods that        reduces their palatability.    -   Low water binding capacity: the starch possesses lower water        holding capacity than most other fiber sources, including other        types of resistant starches. It reduces water in the formula,        ideal for targeting crispiness, and improves shelf life        regarding micro-activity and retrogradation.    -   Process tolerant: the starch is stable against energy intensive        procedures, such as extrusion, pressure cooking, etc.    -   Sensory attributes: such as smooth, non-gritty texture, white,        “invisible” fiber source, and neutral in flavor.

Therefore, flour or starch produced from the durum wheat of the presentinvention can be used to replace bread wheat flour or starch, to producewheat bread, muffins, buns, pasta, noodles, tortillas, pizza dough,breakfast cereals, cookies, waffles, bagels, biscuits, snack foods,brownies, pretzels, rolls, cakes, and crackers, wherein the foodproducts may have one or more desired features.

In some embodiments, the mutant durum wheat has one or more phenotypeswhen compared to a wild-type durum wheat of the same species, whichincludes, but are not limited to, modified gelatinization temperature(e.g., a modified amylopectin gelatinization peaks, and/or a modifiedenthalpy), modified amylose content, modified resistant amylose content,modified starch quality, modified flour swelling power, modified proteincontent (e.g., higher protein content), modified kernel weight, modifiedkernel hardness, and modified semolina yield.

In some embodiments, the methods relate to modifying gelatinizationtemperature of durum wheat, such as modifying amylopectin gelatinizationpeaks and/or modifying enthalpy. Modified gelatinization temperatureresults in altered temperatures required for cooking starch basedproducts. Different degrees of starch gelatinization impact the level ofresistant starch, or example, endothermic peaks I and II of FIG. 5 aredue to the resolved gelatinization and the melting of the fat/amylosecomplex, respectively. In some embodiments, the amylopectingelatinization profile of the durum wheat of the present invention ischanged compared to reference durum wheat, such as a wild-type durumwheat. In some embodiments, the amylopectin gelatinization temperatureof the durum wheat of the present invention is significantly lower thanthat of a wild-type control. For example, the amylopectin gelatinizationtemperature of the durum wheat of the present invention is about 1° C.,2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 15° C.,20° C., 25° C. or more lower than that of a wild-type control based onpeak height on a Differential Scanning Calorimetry (DSC) thermogram,under the same heating rate. Starches having reduced gelatinization areassociated with those starches having increased amylose and reducedglycemic index. They are also associated with having firmer starch basedgels upon retrogradation as in cooked and cooled pasta.

In some embodiments, the change in enthalpy of the durum wheat starch ofthe present invention is dramatically smaller compared to that of a wildtype control. For example, as measured by DSC thermogram, the heat flowtransfer in the durum wheat starch of the present invention is onlyabout ½, ⅓, or ¼ of that of a wild-type control.

Starch gelatinization is a process that breaks down the intermolecularbonds of starch molecules in the presence of water and heat, allowingthe hydrogen bonding sites (the hydroxyl hydrogen and oxygen) to engagemore water. This irreversibly dissolves the starch granule. Penetrationof water increases randomness in the general starch granule structureand decreases the number and size of crystalline regions. Crystallineregions do not allow water entry. Heat causes such regions to becomediffuse, so that the chains begin to separate into an amorphous form.Under the microscope in polarized light starch loses its birefringenceand its extinction cross. This process is used in cooking to make rouxsauce. The gelatinization temperature of starch depends upon plant typeand the amount of water present, pH, types and concentration of salt,sugar, fat and protein in the recipe, as well as derivatisationtechnology used. The gelatinization temperature depends on the degree ofcross-linking of the amylopectin, and can be modified by geneticmanipulation of starch synthase genes.

In one embodiment, the methods relate to modifying amylose content ofdurum wheat, such as resistant amylose content. Flour with increasedresistant amylose content can be used to make firmer pasta with greaterresistance to overcooking as well as reduced glycemic index andincreased dietary fiber and resistant starch. In some embodiments, theamylose content and/or the resistant amylose content of the durum wheatof the present invention and the products produced from said wheat, ismodified (e.g., increased) by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%/o, 77%/o, 79%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%,160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%,1000% or more compared to that of a wild-type durum wheat.

In some embodiments, the amylose content and/or resistant amylosecontent of the durum wheat of the present invention and productsproduced from said wheat is about 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40/%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 79%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%. Thus, wild type durum wheat analyzed by exemplarymethods described herein, was found to have an amylose content of about38% as compared to a high amylose durum wheat of the invention which wasfound to have significantly more than 38% amylose content including,e.g., about 53% amylose.

In some embodiments, the methods relate to modifying starch quality ofdurum wheat.

In some embodiments, the methods relate to modifying flour swellingpower (FSP) of durum wheat. Reduced FSP should reduced weight of thenoodles and increase firmness. In some embodiments, based on the methodsdescribed in Mukasa et al. (Comparison of flour swelling power andwater-soluble protein content between self-pollinating andcross-pollinating buckwheat, Fagopyrum 22:45-50 (2005), the FSP of thedurum wheat of the present invention is modified (e.g., decreased) byabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 79%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%,400%, 500%, 600%, 700%, 800%, 900%, 1000% or more compared to that of awild-type durum wheat. Flour swelling power may be negatively correlatedwith noodle firmness but positively correlated with cook weight meaningthat as FSP declined noodles were firmer and not as heavy.

In some embodiments, the FSP of the durum wheat of the present inventionand products produced from said wheat is 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0(g/g). Thus, wild type durum wheat analyzed by exemplary methodsdescribed herein, was found to have an FSP of about 8.4 as compared to ahigh amylose durum wheat of the invention which was found to havesignificantly less than 8.4 FSP, including, e.g., about 5.8 FSP.

In some embodiments, the methods relate to modifying amylopectin contentof durum wheat. Amylose and amylopectin are interrelated so decreasingamylopectin is the same benefit as increased amylose. Decreasing amylose(and/or increasing amylopectin) is associated with increased FSP,reduced retrogradation and softer baked products and noodles. Increasingamylopectin is also associated with reduced rate of staling. In someembodiments, the amylopectin content of the durum wheat of the presentinvention is modified (e.g., decreased) by about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,79%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%,150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,900%, 1000% or more compared to that of a wild-type durum wheat.

In some embodiments, the amylopectin content of the durum wheat of thepresent invention and products produced from said wheat is about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 79%, 79%, 800/%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In some embodiments, the methods relate to modifying protein content ofdurum wheat. In some embodiments, the protein content of the durum wheatof the present invention and the products produced from said durumwheat, is modified (e.g., increased) by about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,79%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%,150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,900%, 1000% or more compared to that of a wild-type durum wheat.

In some embodiments, the protein content of the durum wheat of thepresent invention and products produced from said wheat is about 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 79%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

Thus, wild type durum wheat products analyzed by exemplary methodsdescribed herein, was found to have a protein content of about 16.8% ascompared to a high amylose durum wheat product of the invention whichwas found to have significantly more than 16.8% protein content,including, e.g., about 22.8% protein. Increased protein content meansgreater nutritional value (reduced glycemic index) as well as greaterfunctionality. In terms of pasta quality, increased protein contentwould be associated with reduced FSP and increased pasta firmness.

In some embodiments, the methods relate to modifying dietary fibercontent in the durum wheat grain. In some embodiments, the dietary fibercontent in the durum wheat grain of the present invention and theproducts produced from said durum wheat, is modified (e.g., increased)by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 79%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180/%, 190%, 200%,300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more compared to thatof a wild-type durum wheat.

In some embodiments, the dietary content of the durum wheat of thepresent invention and products produced from said wheat is about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 79%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

Thus, wild type durum wheat products analyzed by exemplary methodsdescribed herein, was found to have a dietary fiber content of about 3%as compared to a high amylose durum wheat product of the invention whichwas found to have significantly more than 3% dietary fiber, including.e.g., about 8.6% dietary fiber.

Advantages of consuming products made from grain with increased dietaryfiber include, but are not limited to the production of healthfulcompounds during the fermentation of the fiber, and increased bulk,softened stool, and shortened transit time through the intestinal tract.

In some embodiments, the methods relate to modifying fat content in thedurum wheat grain. In some embodiments, the fat content in the durumwheat grain of the present invention is modified (e.g., increased) byabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 79%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%,401)%, 500%, 6000/%, 701)%, 800%, 900%, 1000% or more compared to thatof a wild-type durum wheat.

In some embodiments, the fat content of the durum wheat of the presentinvention and products produced from said wheat is about 0%, 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.3%, 1.4%, 1.5%,1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%,2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%,4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, or 40%.

Thus, wild type durum wheat products analyzed by exemplary methodsdescribed herein, was found to have a fat content of about 1.9% ascompared to a high amylose durum wheat product of the invention whichwas found to have significantly more than 1.9% fat content, including,e.g., about 3.5% fat.

In some embodiments, the methods relate to modifying resistant starchcontent in the durum wheat grain. In some embodiments, the resistantstarch content in the durum wheat grain of the present invention ismodified (e.g., increased) by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 79%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%,170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%or more compared to that of a wild-type durum wheat.

In some embodiments, the resistant starch content of the durum wheat ofthe present invention and products produced from said wheat is about0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 0.3%,1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%,2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%,3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%,5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 79%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%.

Thus, wild type durum wheat products analyzed by exemplary methodsdescribed herein, was found to have a resistant starch content of about<2% as compared to a high amylose durum wheat product of the inventionwhich was found to have significantly more than <2% resistant starch,including, e.g., about 3.8% resistant starch.

In some embodiments, the methods relate to modifying ash content in thedurum wheat grain. In some embodiments, the ash content in the durumwheat grain of the present invention is modified (e.g., increased) byabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 79%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%,400%, 500%, 600%, 700%, 800%, 900%, 1000% or more compared to that of awild-type durum wheat.

In some embodiments, the ash content of the durum wheat of the presentinvention and products produced from said wheat is about 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.3%, 1.4%, 1.5%,1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%,2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%,4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%,5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%.

Thus, wild type durum wheat products analyzed by exemplary methodsdescribed herein, was found to have an ash content of about 0.7% ascompared to a high amylose durum wheat product of the invention whichwas found to have significantly more than 0.7% ash content, including,e.g., about 1.2% ash.

In some embodiments, the methods relate to modifying kernel weight ofdurum wheat. In some embodiments, the kernel weight of the durum wheatof the present invention is modified (e.g., decreased) by about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 79%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%,130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%,700%, 800%, 900%, 1000% or more compared to that of a wild-type durumwheat. For example, the SGP1 null of the present invention may havereduced kernel weight. Reduced kernel weight is often associated withincreased protein content and its associated benefits as describedabove. Increased seed weight without impacting seed number leads toincreased yield and generally increased starch content.

In some embodiments, the kernel weight of the durum wheat grain of thepresent invention is about 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, or 50 mg.

Thus, wild type durum wheat analyzed by exemplary methods describedherein, was found to have a kernel weight of about 40.3 mg as comparedto a high amylose durum wheat product of the invention which was foundto have significantly less than 40.3 mg kernel weight, including, e.g.,about 34.8 mg.

In some embodiments, the methods relate to modifying kernel hardness ofdurum wheat.

In some embodiments, the kernel hardness of the durum wheat of thepresent invention is modified (e.g., increased or decreased) for about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 79%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%,110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%,500%, 600%, 700%, 800%, 900%, 1000% or more compared to that of awild-type durum wheat.

In some embodiments, the kernel hardness of the durum wheat grain of thepresent invention is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 79,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100.

Thus, wild type durum wheat analyzed by exemplary methods describedherein, was found to have a kernel hardness of about 79 as compared to ahigh amylose durum wheat product of the invention which was found tohave significantly more than 79 kernel hardness, including, e.g., about89.8.

In some embodiments, the kernel hardness is measure by the methodsdescribed in Osborne, B. G., Z. Kotwal, et al. (1997). “Application ofthe Single-Kernel Characterization System to Wheat Receiving Testing andQuality Prediction.” Cereal Chemistry Journal 74(4): 467-470, which isincorporated herein by reference in its entirety. Kernel hardnessimpacts milling properties of wheat. For example, the SGP1 null of thepresent invention may have reduced kernel hardness. Reducing kernelhardness is associated with increased break flour yield and reducedflour ash and starch damage. Milling energy would also be reduced.Increased kernel hardness is associated with increased milling energy,increased starch damage after milling and increased flour particle size.

In some embodiments, the methods relate to modifying semolina yield ofdurum wheat. In some embodiments, the semolina yield of the durum wheatof the present invention is modified (e.g., increased or decreased) forabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 79%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%,400%, 500%, 600%, 700%, 800%, 900%, 1000% or more compared to that of awild-type durum wheat.

In some embodiments, the semolina yield of the durum wheat of thepresent invention and products produced from said wheat is about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 79%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

Thus, wild type durum wheat analyzed by exemplary methods describedherein, was found to have a semolina yield of about 57.9% as compared toa high amylose durum wheat product of the invention which was found tohave significantly less semolina yield, including, e.g., about 56.7%semolina yield.

In some embodiments, mutations in one or more copies of one or morestarch synthesis genes are integrated together to create mutant plantswith double, triple, quadruple etc. mutations. Such mutants can becreated by classic breeding methods.

In some embodiments, mutations described herein can be integrated intowheat species other than durum wheat by classic breeding methods, withor without the help of marker-facilitated gene transfer methods, such asT. aestivum, T. aethiopicum, T. araraticum, T. boeoticum, T. carthlicum,T. compactum, T. dicoccoides. T. dicoccum, T. ispahanicum, T.karamyschevii, T. macha, T. militinae, T. moncoccum, T. polonicum, T.spelia, T. sphaerococcum, T. timopheevii, T. turunicum, T. turgidum, T.urartu. T. vavilovii, and T. zhukovskyi.

In one embodiment, mutants of a starch synthesis gene having mutationsin evolutionarily conserved regions or sites can be used to producedurum wheat plants with improved or altered phenotypes. In oneembodiment, mutants due to nonsense mutation (premature stop codon), canbe used to produce durum wheat plants with improved or alteredphenotypes. In one embodiment, mutants not in evolutionarily conservedregions or sites, can also be used to produce durum wheat plants withimproved or altered phenotypes.

In some other embodiments, mutant starch synthesis genes can beintegrated with other mutant genes and/or transgenes. Based on theteaching of the present invention, one skilled in the art will be ableto pick preferred target genes and decide when disruption oroverexpression is needed to achieve certain goals, such as mutantsand/or transgenes which can generally improve plant health, plantbiomass, plant resistance to biotic and abiotic factors, plant yields,wherein the final preferred fatty acid production is increased. Suchmutants and/or transgenes include, but are not limited to pathogenresistance genes and genes controlling plant traits related to seedyield.

Genes encoding polypeptides that can ultimately affect starch synthesiscan be modulated to achieve a desired starch production. Suchpolypeptides include but are not limited to, soluble starch synthases(SSS), Granule bound starch synthases (GBSS), such as GBSSI, GBSSII,ADP-glucose pyrophosphorylases (AGPases). starch branching enzymes(a.k.a., SBE, such as SBE I and SBE II), starch de-branching enzymes(a.k.a., SDBE), and starch synthases I, II, III, and IV.

The modulation can achieved through breeding methods which integratedesired alleles into a single wheat plant. The desired alleles can beeither naturally occurring ones or created through mutagenesis. In someembodiments, the desired alleles result in increased activity of theencoded polypeptide in a plant cell when compared to a reference allele.For example, the desired alleles can lead to increased polypeptideconcentration in a plant cell, and/or polypeptides having increasedenzymatic activity and/or increased stability compared to a referenceallele. In some embodiments, the desired alleles result in decreasedactivity of the encoded polypeptide in a plant cell when compared to areference allele. For example, the desired alleles can be eithernull-mutation, or encode polypeptides having decreased activity,decreased stability, and/or being wrongfully targeted in a plant cellcompared to a reference allele.

The modulation can also be achieved through introducing a transgene intoa wheat variety, wherein the transgene can either overexpress a gene ofinterest or negatively regulate a gene of interest.

In some embodiments, one or more alleles which result in increasedamylose synthesis are introduced to a wheat plant, such as allelesresulting in modified soluble starch synthase activity or modifiedgranule-bound starch synthase activity. In some embodiments, saidalleles locate in the A genome and/or the B genome of a durum wheat.

In some embodiments, one or more alleles which result in decreasedamylose synthesis are introduced to a wheat plant, such as allelesresulting in modified soluble starch synthase activity or modifiedgranule-bound starch synthase activity. In some embodiments, saidalleles locate in the A genome and/or the B genome of a durum wheat.

In some embodiments, one or more alleles which result in increasedamylopectin synthesis are introduced to a wheat plant, such as allelesresulting in modified SSI, SSII, and/or SSIII activity, modified starchbranching enzyme (e.g., SBEI, SBEIIa and SBEIIb) activity, or modifiedstarch debranching enzyme activity. In some embodiments, said alleleslocate in the A genome and/or the B genome of a durum wheat.

In some embodiments, one or more alleles which result in decreasedamylopectin synthesis are introduced to a wheat plant, such as allelesresulting in modified SSI, SSII, and/or SSIII activity, modified starchbranching enzyme (e.g., SBEI, SBEIIa and SBEIIb) activity, or modifiedstarch debranching enzyme activity. In some embodiments, said alleleslocate in the A genome and/or the B genome of a durum wheat.

Methods of disrupting and/or altering a target gene have been known toone skilled in the art. These methods include, but are not limited to,mutagenesis (e.g., chemical mutagenesis, radiation mutagenesis,transposon mutagenesis, insertional mutagenesis, signature taggedmutagenesis, site-directed mutagenesis, and natural mutagenesis),knock-outs/knock-ins, antisense and RNA interference.

The present invention also provides methods of breeding wheat speciesproducing altered levels of fatty acids in the seed oil and/or meal. Inone embodiment, such methods comprise

i) making a cross between the mutant durum wheat of the presentinvention to a second wheat species to make F1 plants;ii) backcrossing said F1 plants to said second wheat species;iii) repeating backcrossing step until said mutations are integratedinto the genome of said second wheat species. Optionally, such methodcan be facilitated by molecular markers.

The present invention provides methods of breeding species close todurum wheat, wherein said species produces altered/improved starch. Inone embodiment, such methods comprise

i) making a cross between the wheat mutants of the present invention toa species close to durum wheat to make F1 plants;ii) backcrossing said F1 plants to said species that is close to durumwheat:iii) repeating backcrossing step until said mutations are integratedinto the genome of said species that is close to durum wheat. Specialtechniques (e.g., somatic hybridization) may be necessary in order tosuccessfully transfer a gene from durum wheat to another species and/orgenus. Optionally, such method can be facilitated by molecular markers.

The present invention also provides unique starch compositions.

In some embodiments, provided are durum wheat starch compositions havingmodified starch quality compared to the starch compositions derived froma reference durum wheat species, such as a wild-type durum wheatspecies.

In some embodiments, provided are durum wheat starch compositions havingmodified gelatinization temperature compared to the starch compositionsderived from a reference durum wheat species, such as a wild-type durumwheat species. In some embodiments, the durum wheat starch compositionsof the present invention has modified amylopectin gelatinization peaksand/or modified enthalpy. In some embodiments, the amylopectingelatinization temperature of the durum wheat starch of the presentinvention is about 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8°C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17°C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C. ormore higher or lower than that of a wild-type control based on peakheight on a Differential Scanning Calorimetry (DSC) thermogram, underthe same heat rate, or based on a Rapid Visco Analyzer test. Increasedamylose would result in increased gelatinization temperature, thetemperature of amylopectin gelatinization.

Using the methods of the present application, durum wheat grains withbeneficial features can be produced. Such features include but are notlimited to, modified dietary fiber content, modified protein content,modified fat content, modified resistant starch content, modified ashcontent; and modified amylose content. In some embodiments, durum wheatgrains with one or more of the following features compared to the grainmade from a control durum wheat plant are created: (1) increased dietaryfiber content; (2) increased protein content; (3) increased fat content;(4) increased resistance starch content; (5) increased ash content; and(6) increased amylose content. The durum wheat grain with saidbeneficial features can be used to produce food products, such as noodleand pasta.

Plant Transformation

The present provides transgenic wheat plants with one or more modifiedstarch synthesis genes. The modification can be either disruption oroverexpression.

Binary vector suitable for wheat transformation includes, but are notlimited to the vectors described by Zhang et al., 2000 (An efficientwheat transformation procedure: transformed calli with long-termmorphogenic potential for plant regeneration, Plant Cell Reports (2000)19: 241-250), Cheng et al., 1997 (Genetic Transformation of WheatMediated by Agrobacterium tumefaciens. Plant Physiol. (1997) 115:971-980), Abdul et al., (Genetic Transformation of Wheat (Triticumacstivum L): A Review, TGG 2010, Vol. 1, No. 2, pp 1-7), Pastori et al.,2000 (Age dependent transformation frequency in elite wheat varieties,J. Exp. Bot. (2001) 52 (357): 857-863), Jones 2005 (Wheattransformation: current technology and applications to grain developmentand composition, Journal of Cereal Science Volume 41, Issue 2, March2005, Pages 137-147), Galovic et al., 2010 (MATURE EMBRYO-DERIVED WHEATTRANSFORMATION WITH MAJOR STRESS MODULATED ANTIOXIDANT TARGET GENE,Arch. Biol. Sci., Belgrade, 62 (3), 539-546), or similar ones. Wheatplants are transformed by using any method described in the abovereferences.

To construct the transformation vector, the region between the left andright T-DNA borders of a backbone vector is replaced with an expressioncassette consisting of a constitutively expressed selection marker gene(e.g., the NptII kanamycin resistance gene) followed by one or more ofthe expression elements listed in Table 8 operably linked to a reportergene (e.g., GUS or GFP). The final constructs are transferred toAgrobacterium for transformation into wheat plants by any of the methodsdescribed in Zhang et al., 2000, Cheng et al., 1997, Abdul et al.,Pastori et al., 2000, Jones 2005, Galovic et al., 2010, U.S. Pat. No.7,197,9964 or similar ones to generate polynucleotidc::GFP fusions intransgenic plants.

For efficient plant transformation, a selection method must be employedsuch that whole plants are regenerated from a single transformed celland every cell of the transformed plant carries the DNA of interest.These methods can employ positive selection, whereby a foreign gene issupplied to a plant cell that allows it to utilize a substrate presentin the medium that it otherwise could not use, such as mannose or xylose(for example, refer U.S. Pat. No. 5,767,378; U.S. Pat. No. 5,994,629).More typically, however, negative selection is used because it is moreefficient, utilizing selective agents such as herbicides or antibioticsthat either kill or inhibit the growth of nontransformed plant cells andreducing the possibility of chimeras. Resistance genes that areeffective against negative selective agents are provided on theintroduced foreign DNA used for the plant transformation. For example,one of the most popular selective agents used is the antibiotickanamycin, together with the resistance gene neomycin phosphotransferase(nptII), which confers resistance to kanamycin and related antibiotics(see, for example. Messing & Vierra, Gene 19: 259-268 (1982); Bevan etal., Nature 304:184-187 (1983)). However, many different antibiotics andantibiotic resistance genes can be used for transformation purposes(refer U.S. Pat. No. 5,034,322, U.S. Pat. No. 6,174,724 and U.S. Pat.No. 6,255,560). In addition, several herbicides and herbicide resistancegenes have been used for transformation purposes, including the bargene, which confers resistance to the herbicide phosphinothricin (Whiteet al., Nucl Acids Res 18: 1062 (1990), Spencer et al., Theor Appl Genet79: 625-631(1990), U.S. Pat. No. 4,795,855, U.S. Pat. No. 5,378,824 andU.S. Pat. No. 6,107,549). In addition, the dhfr gene, which confersresistance to the anticancer agent methotrexate, has been used forselection (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983).

The expression control elements used to regulate the expression of agiven protein can either be the expression control element that isnormally found associated with the coding sequence (homologousexpression element) or can be a heterologous expression control element.A variety of homologous and heterologous expression control elements areknown in the art and can readily be used to make expression units foruse in the present invention. Transcription initiation regions, forexample, can include any of the various opine initiation regions, suchas octopine, mannopine, nopaline and the like that are found in the Tiplasmids of Agrobacterium tumefaciens. Alternatively, plant viralpromoters can also be used, such as the cauliflower mosaic virus 19S and35S promoters (CaMV 19S and CaMV 35S promoters, respectively) to controlgene expression in a plant (U.S. Pat. Nos. 5,352,605; 5,530,196 and5,858,742 for example). Enhancer sequences derived from the CaMV canalso be utilized (U.S. Pat. Nos. 5,164,316; 5,196,525; 5,322,938;5,530,196; 5,352,605; 5,359,142; and 5,858,742 for example). Lastly,plant promoters such as prolifera promoter, fruit specific promoters,Ap3 promoter, heat shock promoters, seed specific promoters, etc. canalso be used.

Methods of producing transgenic plants are well known to those ofordinary skill in the art. Transgenic plants can now be produced by avariety of different transformation methods including, but not limitedto, electroporation; microinjection; microprojectile bombardment, alsoknown as particle acceleration or biolistic bombardment; viral-mediatedtransformation; and Agrobacterium-mediated transformation. See, forexample, U.S. Pat. Nos. 5,405,765; 5,472,869; 5,538,877; 5,538,880;5,550,318; 5,641,664; 5,736,369 and 5,736369; International PatentApplication Publication Nos. WO2002/038779 and WO/2009/117555; Lu etal., (Plant Cell Reports, 2008, 27:273-278); Watson et al., RecombinantDNA, Scientific American Books (1992); Hinchee et al., Bio/Tech.6:915-922 (1988); McCabe et al., Bio/Tech. 6:923-926 (1988); Toriyama etal., Bio/Tech. 6:1072-1074 (1988); Fromm et al., Bio/Tech. 8:833-839(1990); Mullins et al., Bio/Tech. 8:833-839 (1990); Hici et al., PlantMolecular Biology 35:205-218 (1997); Ishida et al., Nature Biotechnology14:745-750 (1996); Zhang et al., Molecular Biotechnology 8:223-231(1997); Ku et al., Nature Biotechnology 17:76-80 (1999); and, Raineri etal., Bio/Tech. 8:33-38 (1990)), each of which is expressly incorporatedherein by reference in their entirety.

Breeding Methods

Classic breeding methods can be included in the present invention tointroduce one or more mutants of the present invention into other plantvarieties, or other close-related species that are compatible to becrossed with the transgenic plant of the present invention.

Open-Pollinated Populations.

The improvement of open-pollinated populations of such crops as rye,many maizes and sugar beets, herbage grasses, legumes such as alfalfaand clover, and tropical tree crops such as cacao, coconuts, oil palmand some rubber, depends essentially upon changing gene-frequenciestowards fixation of favorable alleles while maintaining a high (but farfrom maximal) degree of heterozygosity. Uniformity in such populationsis impossible and trueness-to-type in an open-pollinated variety is astatistical feature of the population as a whole, not a characteristicof individual plants. Thus, the heterogeneity of open-pollinatedpopulations contrasts with the homogeneity (or virtually so) of inbredlines, clones and hybrids.

Population improvement methods fall naturally into two groups, thosebased on purely phenotypic selection, normally called mass selection,and those based on selection with progeny testing. Interpopulationimprovement utilizes the concept of open breeding populations: allowinggenes to flow from one population to another. Plants in one population(cultivar, strain, ecotype, or any germplasm source) are crossed eithernaturally (e.g., by wind) or by hand or by bees (commonly Apis melliferaL. or Megachile rotundata F.) with plants from other populations.Selection is applied to improve one (or sometimes both) population(s) byisolating plants with desirable traits from both sources.

There are basically two primary methods of open-pollinated populationimprovement. First, there is the situation in which a population ischanged en masse by a chosen selection procedure. The outcome is animproved population that is indefinitely propagable by random-matingwithin itself in isolation. Second, the synthetic variety attains thesame end result as population improvement but is not itself propagableas such; it has to be reconstructed from parental lines or clones. Theseplant breeding procedures for improving open-pollinated populations arewell known to those skilled in the art and comprehensive reviews ofbreeding procedures routinely used for improving cross-pollinated plantsare provided in numerous texts and articles, including: Allard,Principles of Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds,Principles of Crop Improvement, Longman Group Limited (1979); Hallauerand Miranda, Quantitative Genetics in Maize Breeding, Iowa StateUniversity Press (1981); and, Jensen, Plant Breeding Methodology, JohnWiley & Sons, Inc. (1988).

Mass Selection.

In mass selection, desirable individual plants are chosen, harvested,and the seed composited without progeny testing to produce the followinggeneration. Since selection is based on the maternal parent only, andthere is no control over pollination, mass selection amounts to a formof random mating with selection. As stated herein, the purpose of massselection is to increase the proportion of superior genotypes in thepopulation.

Synthetics.

A synthetic variety is produced by crossing inter se a number ofgenotypes selected for good combining ability in all possible hybridcombinations, with subsequent maintenance of the variety by openpollination. Whether parents are (more or less inbred) seed-propagatedlines, as in some sugar beet and beans (Vicia) or clones, as in herbagegrasses, clovers and alfalfa, makes no difference in principle. Parentsare selected on general combining ability, sometimes by test crosses ortopcrosses, more generally by polycrosses. Parental seed lines may bedeliberately inbred (e.g. by selfing or sib crossing). However, even ifthe parents are not deliberately inbred, selection within lines duringline maintenance will ensure that some inbreeding occurs. Clonal parentswill, of course, remain unchanged and highly heterozygous.

Whether a synthetic can go straight from the parental seed productionplot to the farmer or must first undergo one or two cycles ofmultiplication depends on seed production and the scale of demand forseed. In practice, grasses and clovers are generally multiplied once ortwice and are thus considerably removed from the original synthetic.

While mass selection is sometimes used, progeny testing is generallypreferred for polycrosses, because of their operational simplicity andobvious relevance to the objective, namely exploitation of gencralcombining ability in a synthetic.

The number of parental lines or clones that enter a synthetic varieswidely. In practice, numbers of parental lines range from 10 to severalhundred, with 100-200 being the average. Broad based synthetics formedfrom 100 or more clones would be expected to be more stable during seedmultiplication than narrow based synthetics.

Pedigreed Varieties.

A pedigreed variety is a superior genotype developed from selection ofindividual plants out of a segregating population followed bypropagation and seed increase of self pollinated offspring and carefultesting of the genotype over several generations. This is an openpollinated method that works well with naturally self pollinatingspecies. This method can be used in combination with mass selection invariety development. Variations in pedigree and mass selection incombination are the most common methods for generating varieties in selfpollinated crops.

Hybrids.

A hybrid is an individual plant resulting from a cross between parentsof differing genotypes. Commercial hybrids are now used extensively inmany crops, including corn (maize), sorghum, sugarbeet, sunflower andbroccoli. Hybrids can be formed in a number of different ways, includingby crossing two parents directly (single cross hybrids), by crossing asingle cross hybrid with another parent (three-way or triple crosshybrids), or by crossing two different hybrids (four-way or double crosshybrids).

Strictly speaking, most individuals in an out breeding (i.e.,open-pollinated) population are hybrids, but the term is usuallyreserved for cases in which the parents are individuals whose genomesare sufficiently distinct for them to be recognized as different speciesor subspecies. Hybrids may be fertile or sterile depending onqualitative and/or quantitative differences in the genomes of the twoparents. Heterosis, or hybrid vigor, is usually associated withincreased heterozygosity that results in increased vigor of growth,survival, and fertility of hybrids as compared with the parental linesthat were used to form the hybrid. Maximum heterosis is usually achievedby crossing two genetically different, highly inbred lines.

The production of hybrids is a well-developed industry, involving theisolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Crop Plants.

Differential Scanning Calorimetry

Differential scanning calorimetry or DSC is a thermoanalytical techniquein which the difference in the amount of heat required to increase thetemperature of a sample and reference is measured as a function oftemperature. Both the sample and reference are maintained at nearly thesame temperature throughout the experiment. Generally, the temperatureprogram for a DSC analysis is designed such that the sample holdertemperature increases linearly as a function of time. The referencesample should have a well-defined heat capacity over the range oftemperatures to be scanned. DSC can be used to analyze Thermal PhaseChange, Thermal Glass Transition Temperature (Tg), Crystalline MeltTemperature, Endothermic Effects, Exothermic Effects, Thermal Stability,Thermal Formulation Stability, Oxidative Stability Studies, TransitionPhenomena. Solid State Structure, and Diverse Range of Materials. TheDSC thermogram can be used to determine Tg Glass Transition Temperature,Tm Melting point, A Hm Energy Absorbed (joules/gram), Tc CrystallizationPoint, and ΔHc Energy Released (joules/gram).

DSC can be used to measure the gelatinization of starch. See ApplicationBrief, TA No. 6, SII Nanotechnology Inc., “Measurements ofgelatinization of starch by DSC”, 1980; Donovan 1979 Phase transitionsof the starch-water system. Bio-polymers, 18, 263-275.; Donovan, J. W.,& Mapes, C. J. (1980). Multiple phase transitions of starches and Nageliarnylodextrins. Starch, 32, 190-193. Eliasson, A.-C. (1980). Effect ofwater content on the gelatinization of wheat starch. Starch, 32,270-272. Lund, D. B. (1984). Influence of time, temperature, moisture,ingredients and processing conditions on starch gelatinization. CRCCritical Reviews in Food Science and Nutrition, 20 (4), 249-257.Shogren, R. L. (1992). Effect of moisture content on the melting andsubsequent physical aging of cornstarch. Carbohydrate Polymers, 19,83-90. Stevens, D. J., & Elton, G. A. H. (1971). Thermal properties ofthe starch water system. Staerke, 23, 8-11. Wootton, M., &Bamunuarachchi, A. (1980). Application of differential scanningcalorimetry to starch gelatinization. Starch, 32, 126-129. Zobel, H. F.,& Gelation, X. (1984). Gelation. Gelatinization of starch and mechanicalproperties of starch pastes. In R. Whistler, J. N. Bemiller & E. F.Paschall, Starch: chemistry and technology (pp. 285-309). Orlando, Fla.:Academic Press. Gelatinization profile is dependent on heating rates andwater contents. Unless specifically defined, the comparison in DSCbetween the starch from the durum wheat of the present application andthe starch from a wild-type reference durum wheat is under the sameheating rates and/or same water content. In some embodiments, thepresent application provides starch compositions having modifiedgelatinization temperature as measured by DSC.

DSC can be used to measure the glass transition temperature of starch.See Chinachoti, P. (1996). Characterization of thermomechanicalproperties in starch and cereal products. Journal of Thermal Analysis,47, 195-213. Maurice et al. 1985 Polysaccharide-waterinteractions—thermal behavior of rice starch. In D. Simatos & S. L.Multon, Properties of water in foods (pp. 211-227). Dordrecht: Nilhoff.;Slade, L., & Levine, H. (1987). Recent advances in starchretrogradation. In S. S. Stivala, V. Crescenzi & I. C. M. Dea,Industrial polysaccharides (pp. 387-430). New York: Gordon and Breach.Stepto, R. F. T., & Tomka, 1. (1987). Chimia, 41 (3), 76-81. Zeleznak.K. L., & Hoseney, R. C. (1997). The glass transition in starch. CerealChemistry, 64 (2), 121-124. In some embodiments, the present applicationprovides starch compositions having modified glass transitiontemperature as measured by DSC.

DSC can be used to measure the crystallization of starch. SeeBiliaderis, C. G., Page, C. M., Slade, L., & Sirett, R. R. (1985).Thermal behavior of amylose-lipid complexes. Carbohydrate Polymers, 5,367-389. Ring. S. G., Colinna, P., I'Anson, K. J., Kalichevsky. M. T.,Miles, M. J., Morris, V. J., & Orford, P. D. (1987). CarbohydrateResearch, 162, 277-293. In some embodiments, the present applicationprovides starch compositions having modified crystallization temperatureas measured by DSC.

DSC can also be used to calculate the heat capacity change between thestarch made from the durum wheat plants of the present application and awild-type durum wheat plant. The heat capacity of a sample is calculatedfrom the shift in the baseline at the starting transient:

Cp=dH/dt×dt/dT

wherein dH/dt is the shift in the baseline of the thermogram and dt/dTis the inverse of the heating rate. The unit of the heat flow is mW ormeal/second, and the unit of heating rate can be ° C./min or °C./second. In some embodiments, at the heating rate of 10° C./min, theheat capacity of the starch made from the durum wheat of the presentapplication as measured by DSC is modified (e.g., increased ordecreased) for about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 79%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%,190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or morecompared to that of the starch made from a wild-type durum wheat.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing, areincorporated herein by reference.

EXAMPLES Example 1 Impacts of SSII-A Null Allele on Durum Wheat NoodleQuality Materials and Methods

A sample of 200 durum wheat accessions was obtained from the NationalSmall Grains Collection, Aberdeen, Id., and 55 durum wheat accessionswere obtained from the International Center for Agricultural Research inthe Dry Areas (ICARDA). These accessions were screened to identifyaccessions that exhibited a null phenotype for SGP-A1 and/or SGP-B1using SDS-PAGE of starch granule bound proteins.

Starch Extraction

Seeds from a single genotype were ground in a Braun coffee mill (ProctorGamble, Cincinnati. Ohio) for 10 s and then placed in a 2 mlmicrocentrifuge tube along with two 6.5 mm yttria stabilized zirconiaceramic balls (Stanford Materials, Irvine, Calif.) which were thenagitated for 30 s in a Mini-beadbeater-96 (Biospec Products,Bartlesville, Okla.) with an oscillation distance of 3.2 cm and ashaking speed of 36 oscillations/s. The zirconia balls were removed fromthe tubes and 1.0 ml of 0.1 M NaCl was added to the whole grain flourwhich was then left to steep for 30 min. at room temperature. After 30min., a dough ball was made by mixing the wet flour using a plasticKontes Pellet Pestle (Kimble Chase, Vineland, N.J.) and the gluten ballwas removed from the samples after pressing out the starch. The liquidstarch suspension was then transferred to a new pre-weighed 2.0 ml tubeand 0.5 ml ddH₂0 was added to the remnant starch pellet in the firsttube. The first tube was vortexed, left to settle for 1 min. and theliquid starch suspension transferred to the second tube. The starchsuspension containing tubes were centrifuged at 5,000 g and the liquidwas aspirated off. To the starch pellets, 0.5 ml of SDS extractionbuffer (55 mM Tris-Cl pH 6.8, 2.3% SDS, 5% BME, 10% glycerol) was added,the samples were vortexed until suspended, and then centrifuged at 5,000g. The SDS buffer was aspirated off and the SDS buffer extraction wasrepeated once more. Next, 0.5 ml of 80% CsCl was added to the starchpellets, samples were vortexed until suspended, and then centrifuged at7,500 g. The CsCl was aspirated off and the starch pellets were washedtwice with 0.5 ml ddH₂0, and once in acetone with centrifugation speedsof 10,000 g. After aspirating off the acetone the pellets were left todry overnight in a fume hood.

SDS-PAGE of Starch Granule Proteins

To purified starch, 7.5 μl of SDS loading buffer (SDS extraction bufferplus bromophenol blue) was added per milligram of starch. Samples wereheated for 15 min. at 70° C., centrifuged for 1 min at 10,000 g, andthen 40 μl of sample was loaded on a 10% (w/v) acrylamide gel preparedusing a 30% acrylamide/0.8% piperazine diacrylamide w/v stock solution.The gel had a standard 4% w/v acrylamide stacking gel prepared using a30% acrylamide/0.8% piperazine diacrylamide w/v stock solution. Gels(for the mA to be relevant, need the gel length width and height, Andy'spaper lacked that as well.) were run (25 mA/gel for 45 min. and then 35mA/gel for three hrs), silver stained following standard procedures, andphotographed on a light box with a digital camera. Each line wasgenotyped for the presence or absence of the SGP-A1 and/or SGP-B1protein.

Evaluation of Segregating Populations

Two accessions, PI 330546 from NSGC and IG 86304 from ICARDA lacked theSGP-A1 protein. These were both crossed to the adapted durum wheatcultivar ‘Mountrail’ (PVP 990266) (Elias and Miller, 2000). Thepopulations were advanced via single seed descent to the F₅ generation.All lines were genotyped for the presence or absence of the SGP-A1protein using the SDS-PAGE methods described above (FIG. 1). Following ageneration of seed increase, the lines plus parents were evaluated in arandomized block split plot design with two replications. Thepopulations were main plots and the lines within each population weresubplots. Each plot was four 3 m rows spaced 30 cm apart. Plots wereharvested with a plot combine. The trial was grown in separate, adjacentrain fed and irrigated experiments in 2009 and 2010 at the Arthur H.Post Field Research Laboratory near Bozeman, Mont.

Measurement of Grain, Flour and Noodle Characteristics

Flour swelling power (FSP) was measured using seeds from a field grownplot from four replications (two from rain fed and two from irrigatedenvironments) in 2009 and a single replication in 2010. Seeds wereground in a Braun coffee mill (Proctor Gamble, Cincinnati, Ohio) for 10s and then placed in a 2 ml tube along with two 6.5 mm zirconia ballsand then agitated for 30 s in a Mini-beadbeater-96 (Biospec Products,Bartlesville, Okla.) with an oscillation distance of 3.2 cm and ashaking speed of 36 oscillations/s. Next, 30 mg of the whole wheat flourwas weighed out into a 2 ml tube, and 1.5 ml of ddH₂O was added. Sampleswere heated in a Thermomixer® (Eppendorf, Hamburg, Germany) for 30 min.at 92° C. with continuous mixing at 800 rpm. Samples were then cooled onthe bench for 2 min. followed by centrifugation at 4° C./1,000 g for 10min. after which the water was aspirated off. Tubes were then re-weighedand the flour swelling power calculated by dividing the final flourweight by the initial flour weight.

Grain, semolina, and noodle quality characteristics were determined atthe Durum Wheat Quality and Pasta Processing Laboratory, Fargo, N. Dak.Kernel hardness and weight was determined using the Single KernelCharacterization System (SKCS). Kernel protein content and moisturecontent was determined using a Foss Infratec 1241 grain analyzer (FossNorth America, Eden Prairie, Minn.). Kernel weight, grain hardness andgrain protein were measured on all field grown replications from both2009 and 2010.

For the semolina and noodle quality traits, all four field replicationswere measured for the 2010 trial, while grain from the two rain-fed andthe two irrigated replications were composited to form two replicationsfor the 2009 trial. Grain samples were tempered to 15.5% for 24 h andmilled into semolina on a Brabender Quadrumat Jr. mill that is set up tomill durum into semolina. Semolina samples were stored in glass jars at4° C. until used. Semolina protein content and moisture content wasdetermined using a Foss Infratec 1241 grain analyzer. Semolina color wasdetermined by placing semolina in a black holding cell with a quartzglass window, and color was measured with the CIE L, a, b color scaleusing a Minolta CR310 chromameter). L-values measure black to white(0-100); a-values measure redness when positive and greenness whennegative; and b values measure yellowness when positive.

Semolina (75 g) was hydrated to 38% moisture using distilled waterheated to 40° C. Hydration was done in three steps. First, semolina wasmixed for 30 s at low speed using a Kitchen Aid Mixer (model,manufacturer, city, state) while the distilled water was added; second,the mixer was turned off and the hydrated semolina was stirred with aspatula for 30 s, scraping sides of the mixing bowl; and third, thehydrated semolina was mixed with the Kitchen Aid Mixer for 30 s at highspeed. This resulted in crumbly dough that was rounded into a ball,placed in a plastic bag, and rested at room temperature for 20 min. Therested dough was sheeted using the sheeting attachment to the KitchenAid mixer. Three sheeting steps were used, always passing the doughsheet through the machine in the same direction. The sheet was passedthrough the widest roll gap three times, medium roll gap twice, andnarrow roll gap twice. Then the sheet was passed through a fettuccinicutter and laid on trays for drying. The noodles were dried using a lowtemperature (40° C.) drying cycle). During the drying period, relativehumidity of the dryer was decreased from 95% to 50%. The temperature washeld at 40° C. for the first 12 hours, then decreased to 25° C. duringthe last 6 hours of the cycle.

Dried noodles had an average width of 6 mm and thickness of 1.7 mm.Color of dried noodles was measured with a Minolta CR310 chromameter.Noodles were gathered together and measured using a black plasticbackground. Color readings were expressed by Hunter values for L, a, andb. L-values measure black to white (0-100); a-values measure rednesswhen positive and greenness when negative; and b values measureyellowness when positive. Noodles (10 g, 5 cm long) were cooked inboiling distilled water (300 mL) for 18 min. Noodles were drained into aBüchner funnel, rinsed with distilled water (50 mL), and noodles wereweighed. Cooking loss (% total solids weight) was measured byevaporating cooking water to dryness in a forced-air oven at 110° C.Cooked firmness was determined by measuring the work (g.cm) required toshear four cooked noodles using a TX-XT2 texture analyzer (TextureTechnologies Corp., Scarsdale, N.Y.) equipped with a pasta blade. Thefirmness results are an average of four measurements taken for eachcooked sample.

Data Analysis

Because of ample rain fall in both years, the rain-fed and irrigatedtrials were very similar. Therefore, the environment (rain-fed andirrigated)×block combinations were treated as blocks for each year.Analyses of variance combined across years were performed for allmeasured traits using a model for a randomized block split plot combinedover years where the populations were main plots and lines withinpopulations were subplots. Least squares means for each line wereobtained. The subplot and subplot×population sources were partitionedinto SSIIa-A class, line within SSIIa-A class and all possibleinteractions of these sources and with year. Blocks and the lines withina SSIIa-A class were considered random, while all other factors wereconsidered fixed effects. Analyses were performed using the PROC MIXEDprocedure with the SAS/STAT software version 9.3 of the SAS System forWindows (SAS Institute Inc., Cary, N.C.). Differences between SSII-Aclass means for each population were estimated using the ESTIMATEstatement. The lone exception was flour swelling power where the yeareffect was not included in the model. Linear correlations among selectedtraits were obtained using the line means using the PROC CORR procedurewith the SAS/STAT software. The heterogeneity of relationship (slopes)between SSIIa-A allelic classes for specific pairs of variables wastested using methods outlined in Littell et al. p 240 (2002).

Results

Two genotypes were identified that lacked the SGP-A1 protein. These nullgenotype were designated SSIIa-Ab with the wild type designated asSSIIa-Aa. The null genotypes were crossed to Mountrail to createsegregating populations. These segregating populations were evaluated inreplicated trials for two years. The mean grain protein was 14.1% and15.0% for Year 1 and Year 2. Interactions with year were in general notimportant, and data are presented averaged over the two years. TheSSIIa-Ab class had lower FSP than the SSIIa-Ala class (Table I). Thatdifference was larger for the PI 330546 cross than for the IG 86304cross. The SSIIa-Ab class had harder kernels (P<0.05) for both crosses.Kernel weight was lower for the SSIIa-Ab class compared to the SSIIa-Aaclass for the IG 86304 cross. However this difference in kernel weightwas not observed for the PI 330546 cross.

The SSIIa-Ab class had significantly lower semolina yield than SSIIa-Aaclass for the IG 86304 cross (Table 1). Semolina color, measured only in2010, was not significantly affected by SSIIa-A allelic classdifferences. The IG 86304 and PI 330546 parents had lower FSP, higherprotein, lower kernel weight, harder kernels, and lower semolina yieldthan the Mountrail parent (Table 1).

The relative differences between SSIIa-A allelic classes for noodlecolor were similar for the Hunter and CIE color scales (Table 2). TheSSIIa-A allelic difference had negligible effects on noodle colortraits. There was no difference between SSIIa-A allelic classes forresidue or cook weight. The SSIIa-Ab class produced noodles that weremore firm than the SSIIa-Aa class for the PI 330546 cross, but not forthe IG 86304 cross. The result was consistent in both years (data notshown). The IG 86304 and PI 330546 parents produced noodles that weredarker (Lower L) and less yellow (lower b) than the adapted Mountrailparent, both considered undesirable characteristics by consumers. Thesetwo unadapted parents with the SSII-Ab null allele produced noodles thatwere less firm than Mountrail.

Kernel weight was inversely related to grain hardness in both crossesand positively related with semolina yield and noodle firmness for theIG 86304 cross (Table 3). Grain protein was negatively correlated withsemolina yield and FSP in both crosses. Flour swelling power was notstatistically related to any of the noodle quality traits (loss, cookweight or firmness) for the IG 86304 cross, while in the PI 330546 crossFSP was negatively correlated with noodle firmness but positivelycorrelated with cook weight meaning that as FSP declined noodles weremore firm and heavier. The three noodle quality traits, noodle firmness,loss, and cook weight were highly interrelated (Table 3), with loss andcook weight being negatively correlated with firmness and cook weightand cook weight and loss being positively correlated. Theserelationships were consistent between the two crosses.

TABLE 1 Means for grain and semolina traits for two durum wheatrecombinant inbred populations segregating for SSIIa-Aa and SSIIa- Aballeles. Flour Kernel SSIIa-A No swelling Grain weight Grain SemolinaPopulation genotype lines power (g/g) protein % mg hardness^(a) Yield %Semolina L Semolina a Semolina b Mountrail/IG 86304 SSIIa-Aa 25 9.2714.5 37.1 83.7 57.8 82.5 −0.3967 18.85 SSIIa-Ab 10 8.72 14.6 34.8 89.856.7 81.7 −0.1469 19.37 P value^(b) 0.02 0.64 0.02 <0.01 0.03 0.090.2000 0.52 Parents Mountrail 9.70 13.6 40.3 79.0 57.9 84.1 −1.722523.73 IG 86304 8.29 15.0 32.6 94.1 55.7 81.1 0.3211 17.88 Mountrail/PI330546 SSIIa-Aa 22 9.24 14.5 36.5 86.2 57.7 82.0 −0.2194 19.08 SSIIa-Ab24 8.26 14.6 36.6 87.4 57.3 81.6 −0.1596 19.74 P value <0.01 0.93 0.860.38 0.34 0.41 0.6900 0.29 Parents Mountrail 9.36 13.4 40.8 79.5 58.283.5 −1.4525 23.51 PI 330546 7.89 15.2 32.9 95.5 56.1 81.0 0.3900 17.99LSD (0.05)^(c) 0.66 0.3 1.7 2.8 1.3 1.5 0.4640 1.25 ^(a)measured withthe Single Kernel Characterization System. ^(b)P value for comparingSSIIa-Aa vs SSIIa-Ab null class means. ^(c)Compares parent means withina cross.

TABLE 2 Means for noodle color and texture traits for two durum wheatrecombinant inbred populations segregating for SSIIa-Aa and SSIIa-Aballeles. SSII-A No Cooked Firmness Population genotype lines Hunter LHunter a Hunter b CIE L CIE a CIE b Residue g Wt. g g/g Mountrail/IG86304 SSIIa-Aa 25 59.1 2.4343 17.85 65.7 2.8088 25.43 3.83 256.1 22.61SSIIa-Ab 10 57.9 2.9510 17.59 64.6 3.4154 25.29 3.99 251.5 22.71 Pvalue^(a) 0.13 0.09 0.46 0.13 0.09 0.87 0.10 0.19 0.95 Parents Mountrail62.0 0.8867 21.99 68.3 1.0268 32.17 3.90 251.1 24.64 IG 86304 55.63.8948 16.03 62.4 4.5371 23.04 3.96 252.3 21.18 Mountrail/PI 330546SSIIa-Aa 22 59.4 2.4166 17.64 66.0 2.7851 24.99 3.86 248.9 23.75SSIIa-Ab 24 58.8 2.5055 17.97 65.8 2.8919 25.71 3.90 245.2 26.47 P value0.33 0.71 0.72 0.33 0.7 0.27 0.52 0.18 0.04 Parents Mountrail 62.50.9294 22.21 68.8 1.0651 32.41 3.80 247.7 25.30 PI 330546 56.1 3.630016.11 62.9 4.2313 23.07 4.16 247.9 21.67 LSD (0.05)^(b) 1.7 0.3447 0.541.6 0.4085 0.87 0.32 17.3 3.15 ^(a)P value for comparing SSIIa-Aa vs.SSIIa-Ab null class means. ^(b)Compares parent means within a cross.

TABLE 3 Correlations between grain, semolina and noodles quality traitsfor IG86304/Mountrail (upper diagonal) and PI 330546/Mountrail (lowerdiagonal) durum wheat recombinant inbred populations where each issegregating for SSIIa-Aa and SSIIa-Ab alleles. Kernel Grain Flour NoodleCooked Semolina weight hardness Protein swelling firmness Residue Wt.yield Kernel weight 1.00 −0.75^(a) −0.20 −0.13 0.37 −0.17 −0.10 0.41<0.01^(b) 0.25 0.45 0.03 0.32 0.59 0.02 Grain hardness −0.84 1.00 0.21−0.26 −0.34 0.36 0.04 −0.46 <0.01 0.23 0.13 0.04 0.03 0.81 0.01 Protein−0.13 0.77 1.00 −0.42 −0.04 0.06 0.21 −0.69 0.40 0.14 0.01 0.84 0.750.24 <0.01 Flour swelling −0.02 −0.18 −0.39 1.00 −0.24 −0.07 0.19 0.300.89 0.23 0.01 0.17 0.70 −0.28 0.09 Noodle firmness −0.11 0.27 0.47−0.53 1.00 −0.82 −0.79 0.11 0.48 0.07 0.00 0.00 <0.01 <0.01 0.55 Residue0.29 −0.25 −0.25 0.09 −0.66 1.00 0.67 −0.23 0.05 0.09 0.10 0.55 <0.01<0.01 0.18 Cooked Wt. 0.13 −0.26 −0.33 0.52 −0.94 0.67 1.00 −0.04 0.390.08 0.02 0.00 <0.01 <0.01 0.82 Semolina yield 0.07 −0.36 −0.67 0.39−0.38 −0.03 0.29 1.00 0.66 0.01 <0.01 0.01 0.01 0.85 0.05^(a)correlation values are in upper portion of box ^(b)P value fort testof null hypothesis that correlation = 0 are in lower portions of box.

The relationship between FSP and noodle firmness was also examined todetermine if that relationship might differ between SSIIa-A allelicclasses (FIG. 2). The FSP versus noodle firmness relation is homogeneous(slopes are not different) for the PI 330546 cross (P=0.82). Theresponses for the two SSIIa-A classes was also not different for the IG86304 cross (P=0.28). The response equation for FSP versus noodlefirmness for both SSIIa-A classes was ŷ=10.916-0.087x+0.021 (r²=: 0.28)for PI 330546 cross and ŷ=10.010−0.039x+0.028 (r²=0.06) for the IG 86304cross.

Flour swelling power is measured as an indirect measure of amylosecontent in the segregating populations. Flour swelling tests measure theuptake of water during starch gelatinization. There is an inverserelation between flour swelling and amylose content (Crosbie et al.,1992) because of the increased water absorption of amylopectin comparedto amylose (Tester & Morrison, 1990). For example Martin et al. (2004)found negative correlations of r=−0.57 in a bread wheat recombinantinbred population and r=−0.85 in a survey of bread wheat cultivarsbetween amylose content and flour swelling power. Results showed theSSIIa-Ab class had lower swelling power than the SSIIa-Aa class in bothcrosses (Table I). Amylose was not determined in this study. Hogg et al.(2012) determined amylose using differential scanning calorimetry from arandom SSIIa-Aa and SSIIa-Ah null line from the Mountrail/PI 330546cross. They found amylose content was 39.22% for the SSIIa-Ab nullversus 38.02% for the SSIIa-Aa wild type though the difference was notstatistically different (P<0.05). They did find peak amylopectingelatinization temperatures were significantly reduced for the SSIIa-Abnull genotype.

The SSIIa-Ab allele gave lower kernel weight and harder kernels comparedto the SSIIa-Aa allele in the IG 86304 cross (Table I). Kernel weightwas negatively correlated with grain hardness in both crosses meaningsmaller kernels tend to be harder (Table III). The reason for thediffering results for kernel weight and grain hardness between the twocrosses is not clear. The IG 86304 and PI 330546 parents had similarkernel weights and both were significantly less than the Mountrailparent. The PI 330546 cross illustrated that the SSIIa-Abl class noodleswere more firm than their SSII-Aa counterparts (Table II). However therewas no difference in noodle firmness between allele classes for the IG86304 cross even though both crosses had significant difference betweenthe allelic classes in flour swelling. The FSP versus noodle firmnessrelation could not be detected as being different between the SSIIa-Aclasses even though the SSIIa-Ab class for the IG 86304 cross appears torespond differently than the SSIIa-Aa class and the two allelic classesfrom the PI 330546 cross (FIG. 2). One possible explanation might besampling variability resulting from the small number of lines in theSSIIa-Ab null class (10) for the IG 86304 cross. Aside from starchcharacteristics, flour protein may influence noodle texture. In breadwheat increased flour protein leads to firmer noodles (Martin et al.,2010). Protein content does not appear to be a factor in the differingresponse between the two crosses as protein content was nearly the samebetween allelic classes for both crosses.

The SSIIa-A allelic difference was not associated with other changes innoodle quality. This indicates incorporation of the SSIIa-Ab null alleleinto adapted cultivars would not have detrimental effects on noodlequality. One possible advantage of the SSIIa-A4b null allele could bethat the increased noodle firmness from the SSIIa-Ab allele observed inthe PT 330546 may confer increased tolerance to over-cooking. Consumersmay prefer products (noodles or pasta) that are firmer and more tolerantto over-cooking.

Example 2 Creation of a High-Amylose Durum Wheat Through Mutagenesis ofStarch Synthase II

Starch type in cereal seeds is controlled by various starch synthases.The granule bound starch synthase I “Waxy” controls amylose biosynthesiswhile numerous soluble starch synthases are involved in amylopectinbiosynthesis. Mutations in one or more non-granule bound or “soluble”starch synthases lead to decreased amylopectin and increased amylosecontent. Increased amylose in turn is important as it can lower glycemicindex and increase durum (Triticum durum) pasta quality by increasingfirnmess. Here we set out to determine the impact of starch synthase IIa(SSIIa or SGP-1) mutations upon durum starch. As described in Example 1,a screen of durum accessions identified two lines lacking SGP-A1, the Agenome copy of SGP-1. The two lines were determined to carry the sameSGP-A mutation, a 29 bp deletion in the first exon. The SGP-A1 nullswere each crossed with the durum variety ‘Mountrail’ and F₅ derivedSGP-A1 null progeny lines were treated with EMS. From each EMSpopulation, one SGP-B1 null mutation was recovered with each being amissense mutation. Each of the SGP-1 double nulls was found to havelarge increases in amylose content and reduced binding of SGP-2 andSGP-3 to the interior of starch granules. RNA-Seq was used to examinewhat impact the loss of SGP-1 has upon other starch biosynthetic genes.Significant increases in transcript levels of several starchbiosynthetic genes were observed in SGP-1 double nulls relative toMountrail. The resultant high amylose durums may prove useful in thecreation of value added pasta with increased firmness and reducedglycemic index.

Materials and Methods Creation and Screening of a Mutagenized DurumWheat Population

Durum wheat accessions obtained from the USDA National Small GrainsCollection (NSGC, Aberdeen, Id.) and ICARDA were screened for those thatwere null for SGP-A1 and/or SGP-B1 using SDS-PAGE of starch granulebound proteins (see below). From the 200 NSGC Triticum durum corecollection accessions screened, one line, PI-330546, lacked SGP-A1 andnone lacked SGP-B1. From the 55 ICARDA Triticum durum accessionsscreened, one line, IG-86304, lacked SGP-A1 and none lacked SGP-B. Thesetwo lines were crossed independently with the cultivar “Mountrail” (PVP9900266) (Elias and Miller, 2000) and advanced via single seed decent tothe F₅ generation. Lines homozygous for the SGP-A1 null trait that hadseed and plant characteristics similar to Mountrail from each cross werethen treated with ethyl methane sulfonate (EMS) as described in Feiz etal. (2009) with the exception that 0.5% EMS was used and plants wereadvanced two generations in the greenhouse to the M₁:M₂ generation. Seedfrom 294 Mountrail/PI-330546 Mt lines and 196 Mountrail/IG-86304 M₁lines were pre-screened for potential SSIIa-B mutations using a flourswelling power test. For each line, four seeds from a single head wereground in a Braun coffee mill (Proctor Gamble, Cincinnati, Ohio) for 10s and then placed in a 2 ml microcentrifuge tube along with two 6.5 mmyttria stabilized zirconia ceramic balls (Stanford Materials, Irvine,Calif.) and agitated for 30 s in a Mini-beadbeater-96 (Biospec Products,Bartlesville, Okla.) with an oscillation distance of 3.2 cm and ashaking speed of 36 oscillations/s. Next, 30 mg of the whole wheat flourwas weighed out into a 2 ml tube and 1.5 ml of ddH₂O was added. Sampleswere heated in a Thermomixer® (Eppendorf, Hamburg, Germany) for 30 min.at 92° C. with continuous mixing at 800 rpm. Samples were then cooled atroom temperature for 2 min. followed by centrifugation at 4° C./1,000 gfor 10 min. after which the water was aspirated off. Tubes were thenre-weighed and the flour swelling power calculated by dividing the finalflour weight by the initial flour weight.

Starch Extraction

For each selected low FSP genotype along with parental controls fourseeds were ground in a Braun coffee mill (Proctor Gamble, Cincinnati,Ohio) for 10 s and then placed in a 2 ml microcentrifuge tube along withtwo 6.5 mm zirconia balls and agitated for 30 s in a Mini-beadbeater-96.The zirconia balls were removed from the microcentrifuge tubes and 1.0ml of 0.1 M NaCl was added to the whole grain flour which was then leftto steep for 30 min. at room temperature. After 30 min., a dough ballwas made by mixing the wet flour using a plastic Kontes Pellet Pestle(Kimble Chase, Vineland, N.J.) and the gluten ball was removed from thesamples after pressing out the starch. The liquid starch suspension wasthen transferred to a new pre-weighed 2.0 ml tube and 0.5 ml ddH₂0 wasadded to the remnant starch pellet in the first tube. The first tube wasvortexed, left to settle for 1 min. and the liquid starch suspensiontransferred to the second tube. The starch suspension containing tubeswere centrifuged at 5,000 g and the liquid was aspirated off. Next, 0.5ml of SDS extraction buffer (55 mM Tris-Cl pH 6.8, 2.3% SDS, 5% BME, 10%glycerol) was added, the samples were vortexed till suspended, and thencentrifuged at 5,000 g. The SDS buffer was aspirated off and the SDSbuffer extraction was repeated once more. Then, 0.5 ml of 80% CsCl wasadded to the starch pellets, samples were vortexed till suspended, andcentrifuged at 7,500 g. The CsCl was aspirated off and the starchpellets were washed twice with 0.5 ml ddH₂0, and once in acetone withcentrifugation speeds of 10,000 g. After supernatant aspiration thestarch pellets were left to dry overnight in a fume hood.

SDS-PAGE of Starch Granule Proteins

To purified starch, 7.5 μl of SDS loading buffer (SDS extraction bufferplus bromophenol blue) was added per mg of starch. Samples were heatedfor 15 min. at 70° C., centrifuged for 1 min at 10,000 g. and then 40 μlof sample was loaded on a 10% (w/v) acrylamide gel prepared using a 30%acrylamide/0.8% piperazine diacrylamide w/v stock solution. The gel hada standard 4% w/v acrylamide stacking gel prepared using a 30%acrylamide/0.8% piperazine diacrylamide w/v stock solution. Gels wererun (25 mA/gel for 45 min. and then 35 mA/gel for three hrs), silverstained following standard procedures, and photographed on a light boxwith a digital camera.

PCR Screening for Mutations in SSIIa-A and SSIIa-B.

Leaf tissue from M₂ plants suspected of having ssIIa-B mutations andparental lines was collected at Feekes growth stage 1.3, stored at −80°C. and DNA was extracted following Riede and Anderson (1996). Codingregions of SSIIa-A and SSIIa-B were amplified from duplicate DNA samplesusing previously described primers and PCR conditions (Chibbar et al.2005, Shimbata et al. 2005. Sestili et al. 2010a). Amplicons weresequenced at the University of California Berkeley Sequencing Facilityand resultant DNA sequences were analyzed for single nucleotidepolymorphisms using Seqman Pro in the Lasergene 10.1 Suite (DNASTAR,Madison, Wis.). The two durum high amylose (DHA) SGP-1 double mutantsdiscovered were DHA175, from the Mountrail/PI-330546 cross and DHA55,from the Mountrail/IG-86304 cross.

Differential Scanning Calorimetry

For Mountrail, Mountrail/PI 330546 (SGP-A1 null), DHA175 and DHA55differential scanning calorimeter (DSC) analysis was carried out using aPyris 7 Diamond DSC (Perkin Elmer, Norwalk Conn., USA) following themethods described in Hansen et al. (2010). Three biological replicateswere run in triplicate for each genotype. Approximately 10 mg of starch(actual weight was recorded) per sample was placed in a high-pressurestainless steel pan along with 55 μL of ddH₂O. The pan was sealed withan O-ring and cover and the starch was left to hydrate overnight at roomtemperature. Samples were re-weighed the next day then placed at 25° C.for two min to equilibrate before they were heated to 120° C. at 10°C./min. Heat transfer in the samples was compared to an empty stainlesssteel pan as a reference. The Pyris software was used to generatethermograms and calculate transition temperatures and heat of physicaltransition. Amylose was determined via DSC using the methods describedin Polaske et al. (2005). Statistical analysis on amylose content wascarried out using PROC GLM and t-tests with an alpha of 0.05 in SAS 9.0(SAS Institute, Cary, N.C.).

Microscopic Analysis of Starch Granules.

Purified starch granules from Mountrail, Mountrail/PI 330546 (SGP-A1null), DHA175 and DHA55 were obtained from three biological replicatesper sample using the methods described above. Individual starch sampleswere placed on carbon tape which was then sputtered with iridium (20 mAfor 30 s). Starch granules were then observed and photographed using aZeiss Supra 55VP field emission gun-SEM (Carl Zeiss Microscopy, Peabody,Mass.).

Starch Synthesis Gene Expression Analysis Via RNA-Seq

To analyze expression levels of starch synthesis genes, developing seeds14 days post anthesis were collected from Mountrail, DHA55, and DHA175and stored at −80° C. For each genotype, developing seeds were collectedfrom three separate plants, with each plant sample composed of fourseeds from the middle of three different spikes (12 seeds total). Seedswere then ground to a fine powder in liquid N₂ using a pre-chilledmortar and pestle. Total RNA was extracted from immature kernels usingan RNeasy Plant Mini Kit (Qiagen, Valencia, Calif.) after firstpre-extracting each sample to remove excess starch. To accomplish this,one hundred mg of seed powder was transferred to a pre-chilled 1.5 mLtube and 0.5 mL of RNA extraction buffer (100 mM Tris pH 8.0, 150 mMLiCl, 50 mM EDTA, 1.5% (w/v) SDS, 0.15% (v/v) BME) was added andvortexed until homogenous. Next, 0.25 mL of 1:1 (v/v) phenol-chloroform(pH 4.7) was added and samples were mixed by inversion followed by acentrifugation at 13,000×g for 15 min at room temperature. Thesupernatant was transferred to a QIAshredder spin column and total RNAwas extracted per the manufacturer's instructions. Total RNA wasquantified and its quality assessed using a Bioanalyzer (AgilentTechnologies, Santa Clara, Calif.). For RNA-Seq analysis, one μg oftotal RNA was used for the creation of cDNA libraries using TruSeqRNA-Seq library kits (Illumina, San Diego, Calif.) per themanufacturer's instructions. Amplicons from cDNA libraries weresequenced as single 50 bp reads using a LifeTech SOLID 5500xl (LifeTechnologies, Carlsbad, Calif.). RNA-Seq data was analyzed using Q-Seqin ArrayStar v5.0 (DNASTAR, Madison, Wis.). Genes of interest wereselected from the NCBI database for analysis with the match settings inQSeq set to 100% for at least 40 bp with mer minimization turned off.All other settings were left to default and sequences were normalizedusing Reads Per Kilobase of exon model per Million mapped reads (RPKM)method. Resultant linear counts were then further normalized to theexpression levels of the house keeping gene glyceraldehyde-3-phosphatedehydrogenase (Ga3pd). Student's t-tests were used to compare expressionlevels between Mountrail and the two ssIIa null genotypes, DHA55 andDHA175.

Results Screening of EMS Mutagenized Durum Lines

Seed from Mountrail/PI-330546 and Mountrail/IG-86304 M₁ lines wasscreened indirectly for mutations in SSIIa-B using a flour swellingpower test (Table 4). Lines that had a flour swelling power of less than6.5 were selected for analysis of SGPs via SDS-PAGE. One line from theMountrail/PI-330546 cross, DHA175 was lacking SGP-A1/B1, SGP-2 and SGP-3and line DHA55 from the Mountrail/IG-86304 cross had a SGP-B1 band thatwas approximately half the intensity of the Mountrail/IG-86304(wild-type) control (data not shown), indicating a potentialheterozygote. After growing this line another generation (M₂:M₃) it wasconfirmed to be a heterozygote using SDS-PAGE of the SGPs fromindividual plants. Starch granule proteins from Mountrail/PI-330546(wild-type), Mountrail/PI-330546 (SGP-A1 null), DHA175 and a homozygousSGP-1 double null DHA55 were then analyzed via SDS-PAGE using a dilutionseries to examine the effect of the SGP-1 double nulls on the binding ofthe other SGPs (FIG. 3). In both DHA175 and DHA55 the SGP-A1 and SGP-B1bands were completely missing and the SGP-2 and SGP-3 bands had anintensity that was less than 0.0625× the load of the wild-type control.The WX bands appeared normal in both the SGP-1 double null lines. In theSGP-A1 null control none of the SGP bands appeared altered compared tothe wild-type control.

TABLE 4 Screening of EMS-derived lines using flour swelling power.Population n^(‡) FSP (g/g)^(§) Mountrail/PI-330546 F5 (SGP-1 wild-type)24 8.4 (0.10)a Mountrail/PI-330546 F5 (SGP-1A null) 24 7.5 (0.10)b EMSM₁ Mountrail/PI-330546 294 7.3 (0.29)b DHA175^(†) 2 5.8 (0.15)c EMS M₁Mountrail/IG-86304 196 7.7 (0.05)b DHA55^(†) 2 6.4 (0.20)c ^(†)Theselines are SGP-1 double nulls. ^(‡)N = number of lines used in analysis.^(§)FSP = flour swelling power measured on whole seed meal inwater/flour suspension (g) over weight of flour (g). Means followed withthe same letter are not significantly different at P < 0.05 based on aStudents t-test. Standard errors are in ( ).

PCR Screening for Mutations in SSIIa-A and SSIIa-B.

In the parental SGP-A1 null lines PI-330546 and IG-86304 a 29 bpdeletion was discovered in the first exon at position 145-174 using theprimer set Sgp-A1F3/Sgp-A1R3 (Shimbata et al. 2005). In line DHA175 apoint mutation in SSIIa-B was found in the third exon at position 979where a G/C to AT transition occurred using the primer setSgp-B1F1/Sgp-B1R1 (Sestili et al. 2010a). This changed the 327th aminoacid from aspartic acid (GAT) to asparagine (AAT). In line DHA55 a pointmutation was found in SSIIa-B in the eighth exon at position 1,864 usingthe primer set Sgp-B1F2/Sgp-B1R2 (Shimbata et al. 2005). This was also aG/C to A/T transition that resulted in an aspartic acid (GAC) toasparagine (AAC) change in amino acid 622.

Microscopic Analysis

Several images were taken at various magnification levels of each starchsample to try and obtain a representative unbiased starch granule image.In the Mountrail/PI-330546 (wild-type) line the larger A-type granuleswere smooth and lenticular shaped and the smaller B-type granules werespherical and smooth (FIG. 4). In the Mountrail/PI-330546 (SGP-A1 null)line the A-type starch granules had a wide range of minor deformitiesbut appeared to maintain their smoothness and size. The B-type granulesin the SGP-A1 null line were similar to those observed in the wild-typesample (FIG. 4). In the SGP-1 double null lines, DHA175 and DHA55, theA-type granules were deformed and less plump than in the wild-type andSGP-A1 null samples, and had rough or cracked surfaces (FIG. 4). Starchgranule counts were not done but it appeared that the SGP-1 double nulllines had fewer B-type granules which were also deformed and had adented appearance.

Differential Scanning Calorimetry Analysis

The gelatinization properties and amylose content of SGP-1 double nulland control starches was examined using DSC. The combined heat scanthermogram shows there is a clear alteration in the gelatinization ofamylopectin in the SGP-1 double null lines which is represented by thefirst peak observed around 60° C. (FIG. 5). The SGP-1 double null lineshad altered gelatinization properties over the wild type wheat lines.The SGP-1 double null lines had a significantly lower gelatinizationtemperature based on peak height and a dramatically smaller change inenthalpy (FIG. 5, Table 5). These data indicate a disruption inamylopectin synthesis. The second peak around 105° C. which isassociated with amylose gelatinization was similar in shape and sizeacross all samples with the SGP-1 double null lines having coolergelatinization temperatures and larger changes in enthalpy compared tothe controls (FIG. 5, Table 5). Amylose content in the SGP-A1 null linewas unchanged compared to the wild-type control whereas the SGP-1 doublenull lines had significantly higher amylose content (Table 5). In lineDHA175 there was a 41.1% increase in amylose and a 28.6% increase forDHA55.

TABLE 5 Differential scanning calorimetry analysis of SGP-1 double nullstarches. ID Amylose (%)^(†) Peak 1 (° C.)^(†) ΔH1 (J/g)^(†) Peak 2 (°C.)^(†) ΔH2 (J/g)^(†) Wild-type 38.02 (0.6)a 64.4 (0.44) a 8.6 (0.64) a103.8 (0.15) a 4.7 (1.06) b SGP-A1 null 39.22 (2.0)a 62.4 (0.52) b 7.8(0.72) a 102.6 (0.30) b 5.0 (0.42) ab DHA175 53.63 (1.1)b 57.2 (0.34) c2.8 (0.46) b 102.8 (0.35) ab 7.2 (0.25) a DHA55 48.90 (3.2) b 56.2(0.15) c 2.5 (0.80) b 102.0 (0.40) b 6.7 (0.95) ab P value^(‡) 0.0014<0.0001 0.0002 0.0222 0.1180 ^(†)Parameters were, determined fromthermograms using Pyris 7 DSC software. Values are the mean and standarderror ( ) of three biological replicates. Means followed by the sameletter are not significantly different based on LSD, α = 0.05.^(‡)Wild-type SGP-1 and SGP-A1 F₅ null samples came from the crossMountrail/PI-330546. ^(§)ANOVA P-value.Starch Synthesis Gene Expression Analysis with RNA-Seq

To look at the RNA expression levels of genes involved with starchsynthesis in the SGP-1 double null lines RNA-Seq was employed. Data fromthe two SGP-1 double null lines was combined and when compared toMountrail (SGP-1 wild-type) there were several starch synthesis genesthat had significant changes in transcript levels (Table 6). Thedeletion present in SSIIa-A in both DHA175 and DHA55 caused a dramaticreduction in SSIIa-A transcripts (Table 6). Due to the high homology ofthe SSIIa-A and SSIIa-B genes the few number of hits detected forSSIIa-A may have arose from areas where the two genes are 100%identical. To assess this possibility, the SSIIa-A hits were aligned tothe SSIIa-A gene (Genbank:AJ269503) using Seqman NGEN (DNASTAR, Madison,Wis.). Virtually all the 40-50 bp hits aligned to segments of the genewhere base pair differences existed between the two isoforms, indicatingthat these were fragments from SSIIa-A transcripts and not fragments ofSSIIa-B transcripts (data not shown). The two independent pointmutations in SSIIa-B did not produce the same effect as the deletion inSSIa-A, on the contrary there was a significant up regulation of SSIIa-B(Table 6). Significant up regulation of transcripts was also exhibitedfor starch synthesis genes Wx-A1, SsI-1, SheI-A, ShbeIIa-A, SbeIIa-B,SSIII, the large subunit of AGPase, and Pho1. None of the samples showeda significant difference in transcript levels for the selected gluteningenes or any of the housekeeping genes with the exception of Cyp3 (Table6).

TABLE 6 RNA-Seq expression analysis of starch synthesis genes indeveloping seeds from SGP-1 null lines and Mountrail. Genbank SGP-1Null/ Accession Gene Mountrail^(§) DHA55^(§) DHA175^(§) SGP-1 null^(¶)WT^(#) AJ269503 Starch synthase II (Ss2a-A) 876 (57) 75 (22) 44 (12) 59(19) 0.07*** AJ269504 Starch synthase II (Ss2a-B) 1,145 (117) 2,477(370) 2,020 (180) 2,249 (297) 1.96** AB019622‡ Granule-bound starchsyruhase I 4,410 (515) 5,811 (341) 5,723 (348) 5,767 (309) 1.31* (Wx-A1)AB019623‡ Granule-bound starch synthase I 7,180 (811) 8,046 (740) 13,039(763) 10,542 (1,716) 1.47 (Wx-B1) AJ292521 Starch synthase 1 (SsI-1) 827(82) 561 (112) 936 (145) 749 (166) 0.91 AJ292522 Starch synthase 1(SsI-2) 3,158 (141) 4,377 (274) 5,110 (311) 4,744 (350) 1.50** AF286318Starch branching enzyme I-A 7,329 (384) 11,694 (1.137) 14,523 (962)13,109 (1,299) 1.79** (Sbe1-A) HE591389†‡ Starch branching enzyme IIa3,629 (190) 4,699 (472) 5,755 (724) 5,227 (641) 1.44* (Sbe2a-A) AY740401Starch branching enzyme IIa-B 1,690 (104) 2,442 (71) 2,345 (295) 2,393(195) 1.42* (Sbe2a-B) AF258608 Starch synthase III (Ss3) 700 (27) 894(69) 1,036 (84) 965 (82) 1.38* AY044844†‡ Starch Synthase IV (Ss4) 21(7) 37 (7) 47 (14) 42 (10) 1.99 DQ839506 ADP-glucose pyrophosphorylase3,083 (258) 6,237 (315) 6,819 (503) 6,528 (418) 2.12*** large subunit(AgpL) AF244997 ADP glucose pyrophosphorylase 26,631 (3,322) 20,690(4,399) 29,136 (1,234) 24,913 (3,935) 0.94 small subunit (AgpS) AJ301647Isoamylase I (Iso1) 1,730 (74) 2,211 (232) 2,113 (285) 2,162 (235) 1.25EF137375† Limit dextrinase debranching 1,469 (85) 1,416 (180) 2,520(337) 1,968 (424) 1.34 enzyme 1 (Ld1) EU595762 alpha-1,4-glucanphosphorylase 1,654 (88) 2,028 (53) 2,449 (263) 2,239 (216) 1.35* (Pho1)U66376 1,4-alpha-D-glucanotransferase 732 (50) 874 (144) 1,311 (157)1,093 (193) 1.49 JF736013†‡ HMW glutenin subunit 30,040 (3,463) 27,288(6,732) 45,134 (2,445) 36,211 (7,236) 1.21 (Glu-B1 Bx7) HQ619891† LMWglutenin subunit (LMW-5) 675,506 (98,596) 461,181 (29,247) 1,300,483(86,856) 880,832 (271,666) 1.30 AF262983 Cyclophilin A-2 (Cyp2) 2,569(234) 3,183 (368) 2,696 (399) 2,939 (376) 1.14 AF262984 Cyclophilin A-3(Cyp3) 954 (84) 1,311 (169) 2,102 (241) 1,706 (312) 1.79* BK001238†Ribosomal protein L3A-1 2,539 (347) 1,944 (297) 2,450 (182) 2,197 (272)0.87 (Rpl3a-1) DQ489316† GTP-binding protein (Gbp-1) 573 (56) 702 (64)791 (79) 747 (70) 1.30 FN429985 glyceraldehyde-3-phosphate 25,582 25,58225,582 25,582 — dehydrogenase (Ga3pd) JF727656‡ ubiquitin-proteinligase/zinc ion 340 (69) 298 (31) 450 (61) 374 (65) 1.10 binding protein(Zfp-1) U76896 Beta-tubulin 5 (Tubb5) 1,387 (86) 1,727 (102) 1,663 (219)1,695 (154) 1.22 †Tissue of origin was unavailable; all other sequencescame from developing endosperms. ‡Sequences are from genomic DNA withall interons removed; all other sequences were mRNA derived. ^(§)Meanlinear counts and standard errors ( ) from three biological replicatesafter normalization to Ga3pd.

Discussion

Our goal was to develop a high-amylose durum line through themutagenesis of SSIIa (SGP-1). There is little natural variation at thislocus as it is a key starch biosynthetic enzyme and after screening 255Triticum durum accessions we only discovered two lines that were SGP-A1null and none that were SGP-B1 null. Interestingly, the two lines thatwere SGP-A1 null, PI-330546 and IG-86304, carried the same 29 bpdeletion located in the first exon. This deletion seemingly produces anunstable mRNA as there was a significant reduction of its transcriptlevels in the two SGP-1 double null lines. This is not the same deletionthat was reported by Shimbata et al. (2005) for the SGP-A1 mutant inbread wheat (Yamamori and Endo 1996). The two separate point mutationscreated through EMS mutagenesis in SSIIa-B did not produce the sameeffect; in fact the expression of SSIIa-B was significantly higher inthe SGP-1 double null lines compared to the cultivar Mountrail. Neitherof the point mutations in SSIIa-B introduced a stop codon but the changeof the effected amino acids (327 in DHA175 and 622 in DHA55) fromaspartic acid to asparagine clearly affected the stability of theenzyme. It is unknown whether these amino acids are critical for theenzymes activity or if they affect the folding of the protein.

As shown in our previous studies several pleiotropic effects wereobserved as the result of the loss of SSII or SGP-1. Herein wedemonstrate that the SGP-double null lines had significant increases intheir amylose content from 38% to 50% (+12%). The two SGP-1 double nulllines had extremely different amylopectin gelatinization peaks from theSGP-A1 null and wild-type which were characterized by a decreasedenthalpy and reduced gelatinization temperature (FIG. 5, Table 5). Inline DHA55 the peak for amylopectin gelatinization was almost too smallto distinguish. Accordingly, the SGP-1 double null lines also had alower flour swelling power (Table 4). These results are evidence of adisruption in amylopectin synthesis. Both types of starch granules fromSGP-1 double nulls were deformed and had rough or cracked surfaces.While not statistically determined, we observed an overall decrease inthe amount of B-type starch granules in the durum SGP-1 double nulllines. There was an almost complete loss of other starch biosyntheticenzymes from the interior of starch granules, namely SBEII (SGP-2) andSSI (SGP-3), while GBSSI remained intact. The loss of these proteinspresence in the starch granules however did not mean that these proteinswere not produced. It has been shown that in the soluble fraction of theendosperm SBEII, SSI, and GBSSI accumulate at normal levels(Kosar-Hashemi et al. 2007, Morell et al. 2003). It has beenhypothesized that SSs, SBEs, along with other starch biosyntheticenzymes act together in complexes in the wheat amyloplast and when oneof these enzymes is disrupted it has significant effects on the otherenzymes (Tetlow et al. 2004a). In the SGP-1 double null lines, this ismanifested by the lack of entrapment of SSI and SBEII in the starchgranule matrix. Tetlow et al. (2008) demonstrated that in bread wheatSBEII, SSI, and SSIIa interact to form a complex during starchdeposition which is controlled by phosphorylation. The loss of SSIIlikely restricts the formation of this complex and in turn long-chainamylopectin formation and the entrapment of SBEII and SSI.

Using RNA-Seq to analyze the transcript levels of the genes involved instarch synthesis in SGP-1 double null lines there was indeed no negativeeffect on starch synthesis gene expression but in some cases anup-regulation. For Wx-A1, SsI-1, SbeI-A, SbeIIa-A, SbeIIa-B, SSIII, AgpL(large subunit of AGPase), and Pho1 (alpha-1,4-glucan phosphorylase)there was a significant increase in the transcript levels of these genesin the SGP-1 double null lines. In general starch biosynthetic genestrended upward in expression in the SGP-1 double null lines. Theup-regulation of starch biosynthetic genes after the elimination of akey enzyme has also been observed in bread wheat where SbeIIa wassilenced using RNAi (Sestili et al. 2010b). Using qRT-PCT Sestili et al.(2010b) saw increases in Wx-1, SSIII, Iso1, and Ld1 transcripts but noincrease for SsI, SSIIa, SbeIIb, or SbeI. The increase of starchsynthesis related transcripts in the durum SGP-1 double null lines wasmuch more moderate than those observed by Sestili et al. (2010b) and islikely due to the different methodologies used. Quantitative RT-PCRexpression data presents relative differences through fold changeswhereas RNA-seq provides a more precise assessment of transcriptnumbers. This phenomenon of starch biosynthetic genes being up-regulatedwhen one of the critical genes is turned off through mutation or othermeans has yet to be fully explained. It could be that there is negativefeedback that controls the expression of starch synthesis genes and thelack of SSII causes these genes to be up regulated. In SGP-1 mutants inbread wheat (Yamamori et al. 2000) and barley (Morell et al. 2003) itwas noted that there was a significant decrease in starch content whichseems peculiar given this up-regulation of most starch synthesis genes.However, knowing that these enzymes act in coordination it is reasonableto assume that maximum starch content is not achievable when thesecomplexes do not form properly.

Given the high amylose content, altered gelatinization properties, anddecreased flour swelling power of the two durum SGP-1 double-mutantlines presented here it is reasonable to assume that there will besignificant impact on their end use quality. In an experiment wherenoodles were made from the Mountrail/PI-33038 F5 and Mountrail/IG-88905F5 populations there was an increase in noodle firmness that wasassociated with the SGP-A1 null trait. The SPG-1 double null linesshould produce a more profound effect as the amylose content of theSGP-A1 null lines was similar to the wild-type. Along with increasednoodle firmness, there is a possibility that these lines will also havepotential health benefits. In both human and animal trials high amylosebread wheat and barley with increased resistant starch was shown toincrease overall colon health (Bird et al. 2008; Regina et al. 2006) andproduce a lower glycemic index (Halstrom et al. 2011; King et al. 2008).

Example 3 Wheat Breeding Program Using the Durum Wheat Plants HavingModified Starch

Non-limiting methods for wheat breeding and agriculturally importanttraits (e.g., improving wheat yield, biotic stress tolerance, andabiotic stress tolerance etc.) are described in Slafer and Araus, 2007,(“Physiological traits for improving wheat yield under a wide range ofconditions”, Scale and Complexity in Plant Systems Research:Gene-Plant-Crop Relations, 147-156); Reynolds (“Physiological approachesto wheat breeding”, Agriculture and Consumer Protection. Food andAgriculture Organization of the United Nations); Richard et al.,(“Physiological Traits to Improve the Yield of Rainfed Wheat: CanMolecular Genetics Help”, published by International Maize and WheatImprovement Center.): Reynolds et al. (“Evaluating Potential GeneticGains in Wheat Associated with Stress-Adaptive Trait Expression in EliteGenetic Resources under Drought and Heat Stress Crop science”, CropScience 2007 47: Supplement_3: S-172-S-189); Setter et al., (Review ofwheat improvement for waterlogging tolerance in Australia and India: theimportance of anaerobiosis and element toxicities associated withdifferent soils. Annals of Botany, Volume 103(2): 221-235); Foulkes etal., (Major Genetic Changes in Wheat with Potential to Affect DiseaseTolerance. Phytopathology, July, Volume 96, Number 7, Pages 680-688(doi: 10.1094/PHYTO-96-0680); Rosyara et al., 2006 (Yield and yieldcomponents response to defoliation of spring wheat genotypes withdifferent level of resistance to Helminthosporium leaf blight. Journalof Institute of Agriculture and Animal Science 27. 42-48.); U.S. Pat.Nos. 7,652,204, 6,197,518, 7,034,208, 7,528,297, 6,407,311: U.S.Published Patent Application Nos. 20080040826, 20090300783, 20060223707,20110027233, 20080028480, 20090320152, 20090320151; WO/2001/029237A2;WO/2008/025097A1; and WO/2003/057848A2.

A durum wheat plant comprising modified starch or certain allele(s) ofstarch synthesis genes of the present invention can be self-crossed toproduce offspring comprising the same phenotypes.

A durum wheat plant comprising modified starch or certain allele(s) ofstarch synthesis genes of the present invention (“donor plant”) can alsocrossed with another plant (“recipient plant”) to produce a F1 hybridplant. Some of the F1 hybrid plants can be back-crossed to the recipientplant for 1, 2, 3, 4, 5, 6, 7, or more times. After each backcross,seeds are harvested and planted to select plants that comprise modifiedstarch, and preferred traits inherited from the recipient plant. Suchselected plants can be used as either a male or female plant tobackcross with the recipient plant.

Example 4 Further Characterizations Starch Content

The starch content of the SGP-1 double null lines and a wild-typecontrol durum wheat line is measured by one or more methods as describedherein, or those described in Moreels et al. (Measurement of StarchContent of Commercial Starches, Starch 39(12):414-416, 1987) or Chianget al. (Measurement of Total and Gelatinized Starch by Glucoamylase ando-toluidine reagent, Cereal Chem. 54(3):429-435), each of which isincorporated by reference in its entirety. Starch content in the SGP-1double null lines is expected to be slightly reduced compared to that ofthe wild-type control durum wheat line.

Glycemic Index

The glycemic index of the SGP-1 double null lines and a wild-typecontrol durum wheat line is measured by one or more methods as describedherein, or those described in Brouns et al. (Glycemic index methodology,Nutrition Research Reviews, 18(1):145-171, 2005), Wolever et al. (Theglycemic index: methodology and clinical implications, Am. J. Clin.Nutr. 54(5):846-54, 1991), or Goni et al., A starch hydrolysis procedureto estimate glycemic index, Human Study, 17(3):427-437, 1997), each ofwhich is incorporated by reference in its entirety.

The glycemic index, glycaemic index, or GI is the measurement of glucose(blood sugar) level increase from carbohydrate consumption. Glucose hasa glycemic index of 100, by definition, and other foods have a lowerglycemic index. The glycemic index of durum wheat pasta was measured bycalculating the incremental area under the two-hour blood glucoseresponse curve (AUC) following a 12-hour fast and ingestion of 50 g ofavailable carbohydrates of DHA175 or wild-type pasta. The AUC of thetest food is divided by the AUC of the standard (either glucose or whitebread, giving two different definitions) and multiplied by 100. Theaverage GI value is calculated from data collected in 5 human subjects.Both the standard and test food must contain an equal amount ofavailable carbohydrate.

The glycemic index of the DHA175 double null lines was found to be lowercompared to the wild-type control durum wheat line (FIG. 6). Subjectsgiven DHA175 pasta also exhibited plasma glucose curves with lowerglucose peaks and higher sustained glucose levels at 90 and 120 minuteswhen compared to wild time control durum (FIG. 7). These results suggestthat DHA175 pasta has a potential for greater satiety, maintainingelevated glucose levels for longer periods of time. The results alsosuggest the DHA75 pasta could also have health benefits over controldurum wheat pasta by reducing insulin glucose spikes after consumption.Without wishing to be bound by any particular theory, the highersustained levels of DHA175 glucose may be due to the higher proteincontent of the DHA175 noodles. The timing (90-120 minutes) of theincreasing glucose levels in subjects fed DHA175 pasta is consistentwith increases in glucose made from amino acids.

Pasta Quality

Quality of pasta made by the flour of the SGP-1 double null lines and awild-type control durum wheat line is tested by one or more methods asdescribed herein, or those described in Landi (Durum wheat, semolina andpasta quality characteristics for an Italian food company, Cheam-OptionsMediterrancennes, pages 33-42) or Cole (Prediction and measurement ofpasta quality. International Journal of Food Science and Technology,26(2): 133-151, 1991), each of which is incorporated by reference in itsentirety.

Pasta firmness (Hardness, Table 7) and resistance to overcooking aremeasured. Pasta firmness is expected to be dramatically increased andovercooking reduced in the SGP-1 double null lines compared to that ofthe wild-type control durum wheat line.

Other qualitative factors of pasta can also be considered in evaluatingpasta quality, including but not limited to the following: (1) the typeof place of origin of the durum wheat from which the flour is produced;(2) the characteristics of the flour; (3) the manufacturing processes ofkneading, drawing and drying; (4) possible added ingredients; and (5)the hygiene of preservation.

Rapid Visco Analyzer (RVA)

Starch of the SGP-1 double null lines and a wild-type control durumwheat line is tested in a Rapid Visco Analyzer (RVA) by one or moremethods as described herein, or those described in Newport ScientificMethod ST-00 Revision 3 (General Method for Testing Starch in RapidVisco Analyzer, 1998), Ross (Amylose, amylopectin, and amylase: Wheat inthe RVA, Oregon State University, 55^(th) Conference Presentation,2008), Bao et al., (Starch RVA profile parameters of rice are mainlycontrolled by Wx gene, Chinese Science Bulletin, 44(22):2047-2051,1999), Ravi et al., (Use of Rapid Visco Analyzer (RVA) for measuring thepasting characteristics of wheat flour as influenced by additives,Journal of the Science of Food and Agriculture, 79(12):1571-1576, 1999),or Gamel et al. (Application of the Rapid Visco Analyzer (RVA) as anEffective Rheological Tool for Measurement of β-Glucan Viscosity,89(1):52-58, 2012), each of which is incorporated by reference in itsentirety.

The SGP-1 double null lines are expected to have reduced peak viscositycompared to that of the wild-type control durum wheat line.

Resistant Starch

Resistant starch content of the SGP-1 double null lines and a wild-typecontrol durum wheat line is tested by one or more methods as describedherein, or those described in McCleary et al., (Measurement of resistantstarch, J. AOAC Int. 2002, 85(3):665-675), Muir and O'Dea (Measurementof resistant starch: factors affecting the amount of starch escapingdigestion in vitro, Am. J. Clin. Nutr. 56:123-127, 1992). Berry(Resistant starch: Formation and measurement of starch that survivesexhaustive digestion with amylolytic enzymes during the determination ofdietary fibre, Journal of Cereal Science, 4(4):301-314, 1986), Englystet al., (Measurement of resistant starch in vitro and in vivo, BritishJournal of Nutrition, 75(5):749-755, 1996), each of which isincorporated by reference in its entirety.

The SGP-1 double null lines have increased resistant starch compared tothe wild-type control durum wheat line in both dry and cooked pastatrials (Table 8 and Table 9).

Example 5 Noodle Firmness

DHA175 and a wild type sister line were grown in the field. The grainwas cleaned, milled and the resulting semolina was used to preparepasta. The milling and pasta processing procedures were as describedpreviously (Carrera et al. 2007). Briefly, durum was milled to semolinausing a Bühler experimental mill fitted with two Miag laboratory scalepurifiers (Biihler-Miag, Minneapolis, Minn., USA). Hydrated semolina wasextruded under vacuum as spaghetti using a DeMaCo semi-commerciallaboratory extruder (DeFrancisci Machine Corp, Melbourne, Fla., USA).Spaghetti was dried in a laboratory pasta drier (Standard Industries,Fargo, N. Dak., USA) using a low temperature (40° C.) drying cycle.

Pasta textural properties were determined by cooking duplicate samplesof each genotype in boiling deionized water until doneness. Cooking timewas determined to be when each pasta was fully cooked through to thecenter of each piece. The DHA175 line had much reduced cooking timerelative to the wild type pasta. Water absorption is the cooked weightdivided by original dry weight with DHA175 having reduced waterabsorption. Cooking loss was determined by drying the cooking water andrecording the percent solids lost with DHA175 having greater cookingloss. Pasta was allowed to drain and cool for five minutes prior totexture analysis. For texture analysis the TA.XT2 Texture Analyzer(Texture Technologies, Scarsdale, N.Y.) was used with a ¼ inch wide flatprobe used to cut into six cooked pieces of pasta. Pasta firmness(hardness) is the peak force during the first compression of spaghettiby the probe. This parameter is related to sensory bite. The DHA175spaghetti was substantially firmer than the wild type spaghetti. Noodleadhesiveness is the negative force between the first and the second peak(work necessary to overcome the attractive forces between the surface ofthe spaghetti and the surface of the probe), and it is theoreticallyrelated to pasta stickiness to teeth at biting. DHA175 pasta was lessadhesive than the wild type pasta. Pasta cohesiveness and chewiness weremeasured as described in (Epstein et al., 2002). The DHA175 pasta alsoshowed slightly lower cohesiveness with significantly higher chewinessscores (Table 7).

TABLE 7 Pasta Textural Properties Cooking Time Water Absorption CookingLoss (min.) Hardness (g) Adhesiveness Cohesiveness Chewiness (%) (%)DHA175 7:30 2382.85 −1.17 .55 1216.49 52.9 8.6 Standard error 0:10 22.521.17 0.01 43.2 4.3 0.2 Wild Type 8:45 1092.93 −5.36 0.63 670.93 63.3 3.7Standard error 0.10  12.14 0.27 0.001 6.8 2.7 0.1 TTEST P 0.001 0.0010.01 0.000 0.000 0.001 0.01

Example 6 Analysis of Food Product

Pasta made from the grain of the SGP-1 double null genotype DHA175 andits wild type sister line durum wheat (“Wild Type”) was further analyzedto determine their nutrient compositions. DHA175 and the Wild Type bothcame from F5-derived lines from the cross between Mountrail×PI330546.The unmutagenized source seed was designated as the Wild Type and thenthis seed was mutagenized and the resultant SGP-1 double null DHA175 wasrecovered. Both dried pasta and cooked pasta were analyzed.

Table 8 provides the nutrient compositions of dried pasta made fromDHA175 and the wild type. The results show that the dried pasta madefrom the SGP-1 double null genotype DHA175 durum wheat has substantiallymore total dietary fiber (“TDFiber”) (e.g., carbohydrates that are notdigestible) than the dried pasta made from the wild type. Therefore, theproducts made from DHA175 are considered to have more dietary fiber thatthose made from the control durum wheat. In addition, the dried pastamade from DHA175 also has increased fat calorie, increased ash content,increased protein content, increased fat content, and increasedresistant starch content when compared to the control durum wheatvariety.

Similar results were observed when comparing cooked pasta made fromDHA175 and the wild type and are provided in Table 9. The cooked sampleswere flash frozen in liquid nitrogen prior to submitting them to the labfor testing. Flash freezing should have prevented retrogradation.

Without wishing to be bound by any theory, the increased dietary fiber,increased protein and/or increased resistant starch in DHA175 are due toincreased amylose content. Alternatively, the increased protein issimply due to the reduced starch content. Ash is also higher in the highamylose pasta made from the DHA175 durum wheat. The reduced seedplumpness in the DHA175 line makes it more difficult to separateendosperm (having lower ash and fiber) from bran (having higher ash andfiber) in the milling process. Thus, without wishing to be bound by anyparticular theory, the increase in fiber content in the DHA175 line maybe due to a decreased endosperm to bran ratio (shrunken seeds) andreduced milling yield, which contributes to the increased fiber contentin addition to the increased amylose content.

The cooking time for pasta made from the DHA175 durum wheat and the wildtype control durum wheat was also determined. As provided in Table 10,the cooking time is significantly reduced when the pasta was made fromthe DHA175 durum wheat.

TABLE 8 Dry Pasta Available Carbohydrates Calories per 100 g servingMoisture Total Dietary Resistant Carbohydrates sample (%) Total CaloriesFat Calories (%) Ash (%) Protein (%) Fiber (%) Fat (%) Starch (%) (%)DHA175-1 52.3 372.0 32.0 10.2 1.2 22.8 8.6 3.5 3.8 53.7 DHA175-2 63.7371.0 29.0 10.2 1.2 21.7 7.8 3.3 3.2 55.9 Wild Type-1 70.2 365.0 17.010.4 0.7 16.8 3.0 1.9 <2.0 67.2 Wild Type-2 70.3 364.0 15.0 10.5 0.716.9 3.3 1.7 <2.0 67.0 DHA175 Avg 63.0 371.5 30.5 10.2 1.2 22.3 8.2 3.43.5 54.8 Wild Type Avg 70.3 364.5 16.0 10.5 0.7 16.9 3.2 1.8 <2.0 67.1 Pvalue 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.05 0.01

TABLE 9 Cooked Pasta Available Carbohydrates Calories per 100 g servingMoisture Total Dietary Resistant Carbohydrates samples (%) TotalCalories Fat Calories (%) Ash (%) Protein (%) Fiber (%) Fat (%) Starch(%) (%) DHA175-1 30.1 176.0 12.0 57.4 0.4 10.8 6.2 1.4 2.2 23.9 DHA175-231.7 178.0 10.0 56.3 0.4 10.4 5.6 1.1 2.0 26.1 Wild Type-1 29.8 156.08.0 61.9 0.1 7.3 1.6 0.9 <2.0 28.2 Wild Type-2 29.4 150.0 5.0 63.1 0.16.8 1.5 0.5 <2.0 27.9 DHA175 Avg 30.9 177.0 11.0 56.9 0.4 10.6 5.9 1.22.1 25.0 Wild Type Avg 29.6 153.0 6.5 62.5 0.1 7.1 1.6 0.7 <2.0 28.1 Pvalue 0.01 0.01 0.10 0.01 0.01 0.01 0.01 0.06 0.06

TABLE 10 Cooking Time (min.) DHA175 7:30 Standard error 0:10 Wild Type8:45 Standard error 0:10 TTEST P 0.001

Example 7 Segregation of SGP-A1 and SGP-B1 Mutants

SGP-1 double null genotypes DHA175 and DHA55 were each crossed with thewild type varieties ‘Mountrail’ and ‘Divide’ with the wild type varietalparent as female in each cross. ˜150 F2 plants from each of the fourpopulations were genotyped using markers specific to either the SGP-A1or SGP-B1 mutations. Genotypes homozygous for the presence or absence ofthe segregating mutations were found at approximately the expectedMendelian ratio of 1/16 for each of the homozygous classes (Table 10).Genotyping revealed the segregation of SGP-1 mutations such that single(individual) SGP-A1 and single SGP-B1 wheat plants were recovered.

TABLE 10 Segregation of SGP-A1 and SGP-B1 mutations Cross wt/wt mut/wtwt/mut mut/mut Mountrail/DHA55 10 7 6 6 Mountrail/DHA175 8 8 6 9Divide/DHA55 11 12 5 7 Divide/DHA175 14 15 10 10

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materials,similar or equivalent to those described herein, can be used in thepractice or testing of the present invention, the non-limiting exemplarymethods and materials are described herein.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. Nothing herein is to beconstrued as an admission that the present invention is not entitled toantedate such publication by virtue of prior invention.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

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1-55. (canceled)
 56. A method for producing a durum wheat plantcomprising one or more mutant durum starch granule protein-B1 (SGP-B1)allele(s), and one or more mutant durum starch granule protein-A1(SGP-A1) allele(s), the method comprising: a. mutagenizing durum wheatgrain to produce a mutagenized population of grain, wherein the durumwheat grain comprises a previously existing mutant durum SGP-B1 alleleor a previously existing mutant durum SGP-A1 allele prior tomutagenizing, and wherein the previously existing mutant durum SGP-B1allele and the previously existing mutant durum SGP-A1 allele are notderived from a hexaploid wheat; b. growing one or more durum wheatplants from said mutagenized durum wheat grain; c. screening theresulting plants from step (b) for a newly produced mutant durum SGP-B1allele or a newly produced mutant durum SGP-A1 allele that is located ona different genome relative to the previously existing mutant durumSGP-B1 allele or the previously existing mutant durum SGP-A1 allele ofstep (a), wherein both the newly produced and previously existing SGP-B1and SGP-A1 mutant alleles disrupt activity of their encoded proteins asdetermined by SDS PAGE; and d. selecting a durum wheat plant comprisingthe newly produced mutant durum SGP-B1 allele or the newly producedmutant durum SGP-A1 allele; wherein the resulting plant comprises one ormore mutant durum SGP-B1 allele(s) and one or more mutant durum SGP-A1allele(s).
 57. The method of claim 56, wherein the durum wheat graincomprises the previously existing mutant durum SGP-A1 allele, and wherethe previously existing mutant durum SGP-A1 allele of step (a) comprisesa 29 bp deletion as found in PI-330546 wheat line.
 58. The method ofclaim 56, wherein the durum wheat grain comprises the previouslyexisting mutant durum SGP-B1 allele, and where the previously existingmutant durum SGP-B1 allele of step (a) encodes an amino acidsubstitution from aspartic acid to asparagine at an amino acid positioncorresponding to amino acid 327 of SEQ ID No.
 6. 59. The method of claim56, wherein the durum wheat grain comprises the previously existingmutant durum SGP-B1 allele, and where the previously existing mutantdurum SGP-B1 allele of step (a) encodes an amino acid substitution fromaspartic acid to asparagine at an amino acid position corresponding toamino acid 622 of SEQ ID No.
 6. 60. The method of claim 56, comprisingan additional step of crossing the plant from step (d) with itself orwith a second plant for one or more generations to produce a durum wheatplant that is homozygous for the mutant SGP-A1 and SGP-B1 alleles. 61.The method of claim 60, wherein the durum wheat plant that is homozygousfor the SGP-A1 and SGP-B1 mutant alleles produces high amylose grain,and wherein the proportion of amylose in the starch of said high amylosegrain is greater than about 38% as measured by differential scanningcalorimetry analysis.
 62. The method of claim 60, wherein the durumwheat plant that is homozygous for the SGP-A1 and SGP-B1 mutant allelesproduces high amylose grain, and wherein the proportion of amylose inthe starch of said high amylose grain is at least 25% higher than theproportion of amylose in the starch from grain of an appropriate durumwheat reference variety grown under similar field conditions.
 63. Themethod of claim 60, wherein the durum wheat plant that is homozygous forthe SGP-A1 and SGP-B1 mutant alleles produces high amylose grain, andwherein the proportion of amylose in the starch of said high amylosegrain is at least 35% higher than the proportion of amylose in thestarch from grain of an appropriate durum wheat reference variety grownunder similar field conditions.
 64. A method of producing a durum wheatplant comprising one or more durum starch granule protein-B1 null(SGP-B1-null) allele(s), and one or more durum starch granule protein-A1null (SGP-A1-null) allele(s), the method comprising: a. crossing a durumwheat plant comprising one or more SGP-A1-null allele(s) with a seconddurum wheat plant comprising one or more SGP-B1-null allele(s) toproduce F1 progeny plants; and b. selecting for an F1 progeny plantcomprising at least one copy of each of the SGP-A1-null and SGP-B1-nullalleles; wherein none of the SGP-A1-null or SGP-B1-null alleles arederived from hexaploid wheat.
 65. The method of claim 64, wherein atleast one SGP-B1-null allele encodes an amino acid substitution fromaspartic acid to asparagine at an amino acid position corresponding toamino acid 327 of SEQ ID No.
 6. 66. The method of claim 64, wherein atleast one SGP-B1-null allele encodes an amino acid substitution fromaspartic acid to asparagine at an amino acid position corresponding toamino acid 622 of SEQ ID No.
 6. 67. The method of claim 64, wherein atleast one SGP-A1-null allele comprises a 29 bp deletion as found inPI-330546 wheat line.
 68. The method of claim 64, comprising anadditional step of crossing the plant from step (b) with itself or witha second plant for one or more generations to produce a durum wheatplant that is homozygous for the SGP-A1-null and SGP-B1-null alleles.69. The method of claim 68, wherein the durum wheat plant that ishomozygous for the SGP-A1-null and SGP-B1-null alleles produces highamylose grain, and wherein the proportion of amylose in the starch ofsaid high amylose grain is greater than about 38% as measured bydifferential scanning calorimetry analysis.
 70. The method of claim 68,wherein the durum wheat plant that is homozygous for the SGP-A1-null andSGP-B1-null alleles produces high amylose grain, and wherein theproportion of amylose in the starch of said high amylose grain is atleast 25% higher than the proportion of amylose in the starch from grainof an appropriate durum wheat reference variety grown under similarfield conditions.
 71. The method of claim 68, wherein the durum wheatplant that is homozygous for the SGP-A1-null and SGP-B1-null allelesproduces high amylose grain, and wherein the proportion of amylose inthe starch of said high amylose grain is at least 35% higher than theproportion of amylose in the starch from grain of an appropriate durumwheat reference variety grown under similar field conditions.
 72. Amethod for producing a durum wheat plant comprising one or more mutantdurum starch granule protein-B1 (SGP-B1) allele(s), and one or moremutant durum starch granule protein-A1 (SGP-A1) allele(s), the methodcomprising: a. mutagenizing durum wheat grain to produce a mutagenizedpopulation of grain; b. growing one or more durum wheat plants from saidmutagenized durum wheat grain; c. screening the resulting plants fromstep (b) for a mutant durum SGP-B1 allele and a mutant durum SGP-A1allele wherein the mutant durum SGP-B1 allele and the mutant durumSGP-A1 mutant alleles disrupt activity of their encoded proteins asdetermined by SDS PAGE; and selecting one or more durum wheat plants,wherein each selected plant comprises a mutant durum SGP-B1 allele and amutant durum SGP-A1 allele.